Immune cells defective for SUV39H1

ABSTRACT

The present invention relates to an engineered immune cell defective for Suv39h1. Preferably, said engineered immune cell further comprises a genetically engineered antigen receptor that specifically binds a target antigen. The present invention also relates to a method for obtaining a genetically engineered immune cell comprising a step consisting in inhibiting the expression and/or activity of Suv39h1 in the immune cell; and further optionally comprising a step consisting in introducing in the said immune cell a genetically engineered antigen receptor that specifically binds to a target antigen. The invention also encompasses said engineered immune cell for their use in adoptive therapy, notably for the treatment of cancer.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of International Application No.PCT/EP2018/066387, filed Jun. 20, 2018, which claims the benefit ofpriority to European Patent Application No. 17305757.1, filed Jun. 20,2017, the contents of which are incorporated herein by reference intheir entirety.

FIELD OF THE INVENTION

The present invention relates to the field of adoptive therapy. Thepresent invention provides immune cells defective for Suv39h1 withenhanced survival, reconstitution potential and central memory phenotypeafter adoptive transfer.

INTRODUCTION

Adoptive T cell therapy (ATCT) using T cells armed with recombinant TCell Receptor (TCR) and Chimeric Antigen Receptor (CAR) technologies isshowing highly encouraging activity in early phase clinical testingagainst several malignancies across a number of Institutions (Kershaw MH, Westwood J A, Darcy P K. Nat Rev Cancer. 2013; 13:525-541).

An emerging theme is that efficient engraftment and long-termpersistence of the therapeutic T cells correlates with positivetherapeutic outcomes. Several pre-clinical studies have shown that naïveand early-differentiated T cells possess an enhanced capacity forlong-term persistence (Berger C et al., J Clin Invest. 2008;118:294-305; Hinrichs C S et al., Proc Natl Acad Sci. 2009;106:17469-17474; Tanel A et al., Expert Rev Vaccines. 2009;8(3):299-312) and can elicit potent anti-tumor responses (Gattinoni L etal., J Clin Investig. 2005; 115:1616-1626; Lugli E et al. J Clin Invest.2013; 123:594-599). Additionally, the increased persistence ofadoptively transferred cells appears to be dependent upon theacquisition of central memory T cell (TCM) populations (Powell D J etal., Blood. 2005; 105(1):241-50; Huang J, Khong H T et al. J Immunother.2005; 28:258-267).

Stable gene transfer has been routinely achieved in the clinical settingnotably through the use of gamma retroviral vectors to transducepolyclonal T cells with CARs (see for example Guest R D et al., CancerImmunol Immunother. 2014; 63:133-145) and TCRs (see notably Johnson L Aet al., Blood. 2009; 114(3):535-46) with these engineered cells showingno obvious adverse safety indications in patients engrafted with CAR Tcells for greater than 10 years (Scholler J et al., Sci Transl Med.2012; 4:132ra153).

For efficient transduction with retroviral vectors or lentiviral,primary T cells need to be actively proliferating (Stacchini A et al.,Leuk Res. 1999; 23:127-136), which is generally achieved through themitogenic stimulation of resting primary T cells.

However, upon activation, T cells progress in an irreversible linearfashion towards an effector (TE) phenotype (Mahnke Y D et al., Eur JImmunol. 2013; 43:2797-2809; Farber D L. Semin Immunol. 2009; 21:84-91).Mitogenic activation for retroviral or lentiviral transduction,therefore, drives differentiation of T cells from a naïve towards a TEphenotype. In combination with ex-vivo culture protocols to expandtransduced T cell numbers to those required for clinical application(about 10⁹-10¹¹), T cells are driven towards a more differentiatedphenotype, which is sub-optimal for systemic persistence.

Thus, while adoptive T cell therapy, including CAR T cell-based therapy,have known remarkable therapeutic successes notably in the treatment ofcertain hematological cancers in the past few years, efficiency has onlybeen shown in a minority of blood cancer types and a few solid tumortypes. It has been hypothesized that low efficacy of the treatments mayresult from limited T cells survival after adoptive transfer.

Therefore, there remains a need in the art for modified or engineered Tcells exhibiting enhanced central memory phenotype and enhanced survivalafter adoptive transfer In particular, there remains a need forproviding immune cells, notably T cells, usable for adoptive therapy,which notably support efficient and broad scale cancer treatment.

SUMMARY OF THE INVENTION

The inventors have now surprisingly discovered that T cells defectivefor Suv39h1 bear an enhanced central memory phenotype and enhancedsurvival after adoptive transfer.

In particular, the inventors showed that T cells defective for Suv39h1accumulate and re-program with increased efficiency into longed-livedcentral memory T cells expressing both CD44 and CD62L. Therefore, thepresent invention relates to modified, or engineered, immune cells,notably modified T cells, wherein Suv39h1 is inactivated.

Said modified, or engineered, immune cells are therefore of highinterest for their use in adoptive therapy. Thus, the present inventionmore specifically relates to an engineered, or modified, immune celldefective for Suv39h1, wherein immune cell preferably further comprisesa genetically engineered antigen receptor that specifically binds atarget antigen.

Typically, the engineered immune cell of claim 1 is a T cell or an NKcell, notably a CD4+ or CD8+ T cell. Preferred cells may be selectedfrom T_(N) cells, TSC_(M), TC_(M) or TE_(M) cells and combinationthereof.

Typically also the engineered immune cell is isolated from a subject.Preferably, said subject is suffering from a cancer, or is at risk ofsuffering from a cancer.

The target antigen to which the genetically engineered antigen receptorspecifically binds is preferably expressed on cancer cells and/or is auniversal tumor antigen.

The genetically engineered antigen receptor can be a chimeric antigenreceptor (CAR) comprising an extracellular antigen-recognition domainthat specifically binds to the target antigen. The geneticallyengineered antigen receptor can also be a T cell receptor (TCR).

Preferably, the activity and/or expression of Suv39h1 in the saidengineered immune cell is selectively inhibited or blocked. In oneembodiment, said engineered immune cell expresses a Suv39h1 nucleic acidencoding a non-functional Suv39h1 protein.

The present invention also relates to a method of producing agenetically engineered immune cell comprising a step consisting ininhibiting the expression and/or activity of Suv39h1 in the immune cell;and optionally a step consisting in introducing into an immune cell agenetically engineered antigen receptor that specifically binds to atarget antigen.

Preferably, the inhibition of Suv39h1 activity and/or expressioncomprises contacting, or putting in contact, the cell with at least anagent inhibiting the expression and/or activity of Suv39h1 and/ordisruption the Suv39h1 gene. Said agent can be selected from smallmolecule inhibitors; antibodies derivatives, aptamers, nucleic acidmolecules that block transcription or translation, or gene editingagents.

The present invention also refers to an engineered immune cell asdescribed herein, or a composition comprising said engineered immunecell, for use in adoptive cellular therapy, notably adoptive therapy ofcancer.

DETAILED DESCRIPTION Definitions

The term “antibody” herein is used in the broadest sense and includespolyclonal and monoclonal antibodies, including intact antibodies andfunctional (antigen-binding) antibody fragments, including fragmentantigen binding (Fab) fragments, F(ab′)2 fragments, Fab′ fragments, Fvfragments, recombinant IgG (rIgG) fragments, variable heavy chain (VH)regions capable of specifically binding the antigen, single chainantibody fragments, including single chain variable fragments (scFv),and single domain antibodies (e.g., sdAb, sdFv, nanobody) fragments. Theterm encompasses genetically engineered and/or otherwise modified formsof immunoglobulins, such as intrabodies, peptibodies, chimericantibodies, fully human antibodies, humanized antibodies, andheteroconjugate antibodies, multispecific, e.g., bispecific, antibodies,diabodies, triabodies, and tetrabodies, tandem di-scFv, tandem tri-scFv.Unless otherwise stated, the term “antibody” should be understood toencompass functional antibody fragments thereof. The term alsoencompasses intact or full-length antibodies, including antibodies ofany class or sub-class, including IgG and sub-classes thereof, IgM, IgE,IgA, and IgD.

An “antibody fragment” refers to a molecule other than an intactantibody that comprises a portion of an intact antibody that binds theantigen to which the intact antibody binds. Examples of antibodyfragments include but are not limited to Fv, Fab, Fab′, Fab′-SH,F(ab′)2; diabodies; linear antibodies; variable heavy chain (VH)regions, single-chain antibody molecules such as scFvs and single-domainVH single antibodies; and multispecific antibodies formed from antibodyfragments. In particular embodiments, the antibodies are single-chainantibody fragments comprising a variable heavy chain region and/or avariable light chain region, such as scFvs.

“Single-domain antibodies” are antibody fragments comprising all or aportion of the heavy chain variable domain or all or a portion of thelight chain variable domain of an antibody. In certain embodiments, asingle-domain antibody is a human single-domain antibody.

As used herein, “repression” of gene expression refers to theelimination or reduction of expression of one or more gene productsencoded by the subject gene in a cell, compared to the level ofexpression of the gene product in the absence of the repression.Exemplary gene products include mRNA and protein products encoded by thegene. Repression in some cases is transient or reversible and in othercases is permanent. Repression in some cases is of a functional orfull-length protein or mRNA, despite the fact that a truncated ornon-functional product may be produced. In some embodiments herein, geneactivity or function, as opposed to expression, is repressed.

Gene repression is generally induced by artificial methods, i.e., byaddition or introduction of a compound, molecule, complex, orcomposition, and/or by disruption of nucleic acid of or associated withthe gene, such as at the DNA level. Exemplary methods for generepression include gene silencing, knockdown, knockout, and/or genedisruption techniques, such as gene editing. Examples include antisensetechnology, such as RNAi, siRNA, shRNA, and/or ribozymes, whichgenerally result in transient reduction of expression, as well as geneediting techniques which result in targeted gene inactivation ordisruption, e.g., by induction of breaks and/or homologousrecombination.

As used herein, a “disruption” of a gene refers to a change in thesequence of the gene, at the DNA level. Examples include insertions,mutations, and deletions. The disruptions typically result in therepression and/or complete absence of expression of a normal or “wildtype” product encoded by the gene. Exemplary of such gene disruptionsare insertions, frameshift and missense mutations, deletions, knock-in,and knock-out of the gene or part of the gene, including deletions ofthe entire gene. Such disruptions can occur in the coding region, e.g.,in one or more exons, resulting in the inability to produce afull-length product, functional product, or any product, such as byinsertion of a stop codon. Such disruptions may also occur bydisruptions in the promoter or enhancer or other region affectingactivation of transcription, so as to prevent transcription of the gene.Gene disruptions include gene targeting, including targeted geneinactivation by homologous recombination.

Cells of the Invention

The cells according to the invention are typically eukaryotic cells,such as mammalian cells (also named in the present invention animalcells), e.g., human cells.

More particularly, the cells of the invention are derived from theblood, bone marrow, lymph, or lymphoid organs (notably the thymus) andare cells of the immune system (i.e., immune cells), such as cells ofthe innate or adaptive immunity, e.g., myeloid or lymphoid cells,including lymphocytes, typically T cells and/or NK cells.

Preferably according to the invention, cells are notably lymphocytesincluding T cells, B cells and NK cells.

Cells according to the invention may also be immune cell progenitors,such as lymphoid progenitors and more preferably T cell progenitors.

T cell progenitors typically express a set of consensus markersincluding CD44, CD117, CD135, and Sca-1 but see also Petrie H T, KincadeP W. Many roads, one destination for T cell progenitors. The Journal ofExperimental Medicine. 2005; 202(1):11-13.

The cells typically are primary cells, such as those isolated directlyfrom a subject and/or isolated from a subject and frozen.

With reference to the subject to be treated, the cells of the inventionmay be allogeneic and/or autologous.

In some embodiments, the cells include one or more subsets of T cells orother cell types, such as whole T cell populations, CD4+ cells, CD8+cells, and subpopulations thereof, such as those defined by function,activation state, maturity, potential for differentiation, expansion,recirculation, localization, and/or persistence capacities,antigen-specificity, type of antigen receptor, presence in a particularorgan or compartment, marker or cytokine secretion profile, and/ordegree of differentiation.

Among the sub-types and subpopulations of T cells and/or of CD4+ and/orof CD8+ T cells are naive T (T_(N)) cells, effector T cells (T_(EFF)),memory T cells and sub-types thereof, such as stem cell memory T(TSC_(M)), central memory T (TC_(M)), effector memory T (T_(EM)), orterminally differentiated effector memory T cells, tumor-infiltratinglymphocytes (TIL), immature T cells, mature T cells, helper T cells,cytotoxic T cells, mucosa-associated invariant T (MAIT) cells, naturallyoccurring and adaptive regulatory T (Treg) cells, helper T cells, suchas TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells,follicular helper T cells, alpha/beta T cells, and delta/gamma T cells.Preferably, the cells according to the invention are T_(EFF) cells withstem/memory properties and higher reconstitution capacity due to theinhibition of Suv39h1, as well as T_(N) cells, TSC_(M), TC_(M), TE_(M)cells and combinations thereof.

In some embodiments, one or more of the T cell populations is enrichedfor, or depleted of, cells that are positive for or express high levelsof one or more particular markers, such as surface markers, or that arenegative for or express relatively low levels of one or more markers. Insome cases, such markers are those that are absent or expressed atrelatively low levels on certain populations of T cells (such asnon-memory cells) but are present or expressed at relatively higherlevels on certain other populations of T cells (such as memory cells).In one embodiment, the cells (such as the CD8⁺ cells or the T cells,e.g., CD3⁺ cells) are enriched for (i.e., positively selected for) cellsthat are positive or expressing high surface levels of CD117, CD135,CD45RO, CCR7, CD28, CD27, CD44, CD127, and/or CD62L and/or depleted of(e.g., negatively selected for) cells that are positive for or expresshigh surface levels of CD45RA. In some embodiments, cells are enrichedfor or depleted of cells positive or expressing high surface levels ofCD122, CD95, CD25, CD27, and/or IL7-Ra (CD127). In some examples, CD8+ Tcells are enriched for cells positive for CD45RO (or negative forCD45RA) and for CD62L.

For example according to the invention, the cells can include a CD4+ Tcell population and/or a CD8+ T cell sub-population, e.g., asub-population enriched for central memory (T_(CM)) cells.Alternatively, the cells can be other types of lymphocytes, includingnatural killer (NK) cells, MAIT cells, Innate Lymphoid Cells (ILCs) andB cells.

The cells and compositions containing the cells for engineeringaccording to the invention are isolated from a sample, notably abiological sample, e.g., obtained from or derived from a subject.Typically the subject is in need of a cell therapy (adoptive celltherapy) and/or is the one who will receive the cell therapy. Thesubject is preferably a mammal, notably a human. In one embodiment ofthe invention, the subject have a cancer.

The samples include tissue, fluid, and other samples taken directly fromthe subject, as well as samples resulting from one or more processingsteps, such as separation, centrifugation, genetic engineering (forexample transduction with viral vector), washing, and/or incubation. Thebiological sample can be a sample obtained directly from a biologicalsource or a sample that is processed. Biological samples include, butare not limited to, body fluids, such as blood, plasma, serum,cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organsamples, including processed samples derived therefrom. Preferably, thesample from which the cells are derived or isolated is blood or ablood-derived sample, or is or is derived from an apheresis orleukapheresis product. Exemplary samples include whole blood, peripheralblood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissuebiopsy, tumor, leukemia, lymphoma, lymph node, gut associated lymphoidtissue, mucosa associated lymphoid tissue, spleen, other lymphoidtissues, and/or cells derived therefrom. Samples include, in the contextof cell therapy (typically adoptive cell therapy) samples fromautologous and allogeneic sources.

In some embodiments, the cells are derived from cell lines, e.g., T celllines. The cells can also be obtained from a xenogeneic source, such asa mouse, a rat, a non-human primate, or a pig. Preferably, the cells arehuman cells.

Cells Defective for Suv39h1

As used herein the term “Suv39h1” or “H3K9-histone methyltransferaseSuv39h1” has its general meaning in the art and refers to the histonemethyltransferase “suppressor of variegation 3-9 homolog 1 (Drosophila)”that specifically trimethylates the Lys-9 residue of histone H3 usingmonomethylated H3-Lys-9 as substrate (see also Aagaard L, Laible G,Selenko P, Schmid M, Dorn R, Schotta G, Kuhfittig S, Wolf A, LebersorgerA, Singh P B, Reuter G, Jenuwein T (June 1999). “Functional mammalianhomologues of the Drosophila PEV-modifier Su(var)3-9 encodecentromere-associated proteins which complex with the heterochromatincomponent M3 1”. EMBO J 1 8 (7): 1923-38.). Said histonemethyltransferase is also known as MG44, KMT1A, SUV39H, SUV39H1,histone-lysine N-methyltransferase SUV39H1, H3-K9-HMTase 1,OTTHUMP00000024298, Su(var)3-9 homolog 1, lysine N-methyltransferase 1A,histone H3-K9 methyltransferase 1, position-effect variegation 3-9homolog, histone-lysine N-methyltransferase, or H3 lysine-9 specific 1.The human Suv39h1 methyltransferase is referenced 043463 in UNIPROT andis encoded by the gene Suv39h1 located on chromosome x (gene ID: 6839 inNCBI) The term Suv39h1 according to the invention also encompasses allorthologs of SUV39H1 such as SU(VAR)3-9.

As used herein the expression “defective for Suv39h1” according to thepresent invention refers to the inhibition, or blockade of Suv39h1activity (i.e., the methylation of Lys-9 of histone H3 by H3K9-histonemethyltransferase) in the cell according to the invention.

“Inhibition of Suv39h1 activity” as per the invention refers to adecrease of Suv39h1 activity of at least 30%, 40%, 50%, 60%, 70%, 80%,90%, 95% or 99% or more as compared to the activity or level of theSuv39h1 protein which is not inhibited. Preferentially, the inhibitionof Suv39h1 activity leads to the absence in the cell of substantialdetectable activity of Suv39h1.

Inhibition of Suv39h1 activity can also be achieved through repressionof Suv39h1 gene expression or though Suv39h1 gene disruption. Accordingto the invention, said repression reduces expression of Suv39h1 in thecell, notably the immune cell of the invention by at least 50, 60, 70,80, 90, or 95% as to the same cell produced by the method in the absenceof the repression. Gene disruption may also lead to a reduced expressionof the Suv39h1 protein or to the expression of a non-functional Suv39h1protein.

By “non-functional” Suv39h1 protein it is herein intended a protein witha reduced activity or a lack of detectable activity as described above.

Thus inhibitors of Suv39h1 activity in a cell according to the inventioncan be selected among any compound or agent natural or not having theability of inhibiting the methylation of Lys-9 of histone H3 byH3K9-histone methyltransferase, or inhibiting the H3K9-histonemethyltransferase SUV39H1 gene expression.

Inhibition of Suv39h1 in the immune cell according to the presentinvention can be permanent and irreversible or transient or reversible.Preferably however, Suv39h1 inhibition is permanent and irreversible.Inhibition of Suv39h1 in the cell may be achieved prior or afterinjection of the cell in the targeted patient as described below.

Genetically Engineered Cells According to the Invention

In some embodiments, the cells comprise one or more nucleic acidsintroduced via genetic engineering that encode one or more antigenreceptors.

Typically, the nucleic acids are heterologous, (i.e., for example whichare not ordinarily found in the cell being engineered and/or in theorganism from which such cell is derived). In some embodiments, thenucleic acids are not naturally occurring, including chimericcombinations of nucleic acids encoding various domains from multipledifferent cell types.

Among the antigen receptors as per the invention are geneticallyengineered T cell receptors (TCRs) and components thereof, as well asfunctional non-TCR antigen receptors, such as chimeric antigen receptors(CAR).

Chimeric Antigen Receptors (CARs)

In some embodiments, the engineered antigen receptors comprise chimericantigen receptors (CARs), including activating or stimulatory CARs,costimulatory CARs (see WO2014/055668), and/or inhibitory CARs (iCARs,see Fedorov et al., Sci. Transl. Medicine, 5(215) (December, 2013)).

Chimeric antigen receptors (CARs), (also known as Chimericimmunoreceptors, Chimeric T cell receptors, Artificial T cell receptors)are engineered receptors, which graft an arbitrary specificity onto animmune effector cell (T cell). Typically, these receptors are used tograft the specificity of a monoclonal antibody onto a T cell, withtransfer of their coding sequence facilitated by retroviral vectors.

CARs generally include an extracellular antigen (or ligand) bindingdomain linked to one or more intracellular signaling components, in someaspects via linkers and/or transmembrane domain(s). Such moleculestypically mimic or approximate a signal through a natural antigenreceptor, a signal through such a receptor in combination with acostimulatory receptor, and/or a signal through a costimulatory receptoralone.

In some embodiments, the CAR is constructed with a specificity for aparticular antigen (or marker or ligand), such as an antigen expressedin a particular cell type to be targeted by adoptive therapy, such as acancer marker. Thus, the CAR typically includes in its extracellularportion one or more antigen binding molecules, such as one or moreantigen-binding fragment, domain, or portion, or one or more antibodyvariable domains, and/or antibody molecules.

The moieties used to bind to antigen fall in three general categories,either single-chain antibody fragments (scFvs) derived from antibodies,Fab's selected from libraries, or natural ligands that engage theircognate receptor (for the first generation CARs). Successful examples ineach of these categories are notably reported in Sadelain M, BrentjensR, Riviere I. The basic principles of chimeric antigen receptor (CAR)design. Cancer discovery. 2013; 3(4):388-398 (see notably table 1) andare included in the present application. scFv's derived from murineimmunoglobulins are commonly used, as they are easily derived fromwell-characterized monoclonal antibodies.

Typically, the CAR includes an antigen-binding portion or portions of anantibody molecule, such as a single-chain antibody fragment (scFv)derived from the variable heavy (VH) and variable light (VL) chains of amonoclonal antibody (mAb).

In some embodiments, the CAR comprises an antibody heavy chain domainthat specifically binds the antigen, such as a cancer marker or cellsurface antigen of a cell or disease to be targeted, such as a tumorcell or a cancer cell, such as any of the target antigens describedherein or known in the art.

In some embodiments, the CAR contains an antibody or an antigen-bindingfragment (e.g. scFv) that specifically recognizes an antigen, such as anintact antigen, expressed on the surface of a cell.

In some embodiments, the CAR contains a TCR-like antibody, such as anantibody or an antigen-binding fragment (e.g. scFv) that specificallyrecognizes an intracellular antigen, such as a tumor-associated antigen,presented on the cell surface as a MHC-peptide complex. In someembodiments, an antibody or antigen-binding portion thereof thatrecognizes an MHC-peptide complex can be expressed on cells as part of arecombinant receptor, such as an antigen receptor. Among the antigenreceptors are functional non-TCR antigen receptors, such as chimericantigen receptors (CARs). Generally, a CAR containing an antibody orantigen-binding fragment that exhibits TCR-like specificity directedagainst peptide-MHC complexes also may be referred to as a TCR-like CAR.

In some aspects, the antigen-specific binding, or recognition componentis linked to one or more transmembrane and intracellular signalingdomains. In some embodiments, the CAR includes a transmembrane domainfused to the extracellular domain of the CAR. In one embodiment, thetransmembrane domain that is naturally associated with one of thedomains in the CAR is used. In some instances, the transmembrane domainis selected or modified by amino acid substitution to avoid binding ofsuch domains to the transmembrane domains of the same or differentsurface membrane proteins to minimize interactions with other members ofthe receptor complex.

The transmembrane domain in some embodiments is derived either from anatural or from a synthetic source. Where the source is natural, thedomain can be derived from any membrane-bound or transmembrane protein.Transmembrane regions include those derived from (i.e. comprise at leastthe transmembrane region(s) of) the alpha, beta or zeta chain of theT-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CDS, CD9, CD 16,CD22, CD33, CD37, CD64, CD80, CD86, CD 134, CD137, CD 154). Thetransmembrane domain can also be synthetic.

In some embodiments, a short oligo- or polypeptide linker, for example,a linker of between 2 and 10 amino acids in length, is present and formsa linkage between the transmembrane domain and the cytoplasmic signalingdomain of the CAR.

The CAR generally includes at least one intracellular signalingcomponent or components. First generation CARs typically had theintracellular domain from the CD3 ζ-chain, which is the primarytransmitter of signals from endogenous TCRs. Second generation CARstypically further comprise intracellular signaling domains from variouscostimulatory protein receptors (e.g., CD28, 41 BB, ICOS) to thecytoplasmic tail of the CAR to provide additional signals to the T cell.Preclinical studies indicated that the second generation improves theantitumor activity of T cells. More recently, third generation CARscombine multiple signaling domains, such as CD3z-CD28-41BB orCD3z-CD28-OX40, to augment potency.

For example, the CAR can include an intracellular component of the TCRcomplex, such as a TCR CD3+ chain that mediates T-cell activation andcytotoxicity, e.g., the CD3 zeta chain. Thus, in some aspects, theantigen binding molecule is linked to one or more cell signalingmodules. In some embodiments, cell signaling modules include CD3transmembrane domain, CD3 intracellular signaling domains, and/or otherCD transmembrane domains. The CAR can also further include a portion ofone or more additional molecules such as Fc receptor γ, CD8, CD4, CD25,or CD16.

In some embodiments, upon ligation of the CAR, the cytoplasmic domain orintracellular signaling domain of the CAR activates at least one of thenormal effector functions or responses of the correspondingnon-engineered immune cell (typically a T cell). For example, the CARcan induce a function of a T cell such as cytolytic activity or T-helperactivity, secretion of cytokines or other factors.

In some embodiments, the intracellular signaling domain(s) include thecytoplasmic sequences of the T cell receptor (TCR), and in some aspectsalso those of co-receptors that in the natural context act in concertwith such receptor to initiate signal transduction following antigenreceptor engagement, and/or any derivative or variant of such molecules,and/or any synthetic sequence that has the same functional capability.

T cell activation is in some aspects described as being mediated by twoclasses of cytoplasmic signaling sequences: those that initiateantigen-dependent primary activation through the TCR (primarycytoplasmic signaling sequences), and those that act in anantigen-independent manner to provide a secondary or co-stimulatorysignal (secondary cytoplasmic signaling sequences). In some aspects, theCAR includes one or both of such signaling components.

In some aspects, the CAR includes a primary cytoplasmic signalingsequence that regulates primary activation of the TCR complex either ina stimulatory way, or in an inhibitory way. Primary cytoplasmicsignaling sequences that act in a stimulatory manner may containsignaling motifs which are known as immunoreceptor tyrosine-basedactivation motifs or ITAMs. Examples of ITAM containing primarycytoplasmic signaling sequences include those derived from TCR zeta, FcRgamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CDS, CD22, CD79a,CD79b, and CD66d. In some embodiments, cytoplasmic signaling molecule(s)in the CAR contain(s) a cytoplasmic signaling domain, portion thereof,or sequence derived from CD3 zeta.

The CAR can also include a signaling domain and/or transmembrane portionof a costimulatory receptor, such as CD28, 4-1BB, OX40, DAP10, and ICOS.In some aspects, the same CAR includes both the activating andcostimulatory components; alternatively, the activating domain isprovided by one CAR whereas the costimulatory component is provided byanother CAR recognizing another antigen.

The CAR or other antigen receptor can also be an inhibitory CAR (e.g.iCAR) and includes intracellular components that dampen or suppress aresponse, such as an immune response. Examples of such intracellularsignaling components are those found on immune checkpoint molecules,including PD-1, CTLA4, LAG3, BTLA, OX2R, TIM-3, TIGIT, LAIR-1, PGE2receptors, EP2/4 Adenosine receptors including A2AR. In some aspects,the engineered cell includes an inhibitory CAR including a signalingdomain of or derived from such an inhibitory molecule, such that itserves to dampen the response of the. Such CARs are used, for example,to reduce the likelihood of off-target effects when the antigenrecognized by the activating receptor, e.g, CAR, is also expressed, ormay also be expressed, on the surface of normal cells.

TCRs

In some embodiments, the genetically engineered antigen receptorsinclude recombinant T cell receptors (TCRs) and/or TCRs cloned fromnaturally occurring T cells.

A “T cell receptor” or “TCR” refers to a molecule that contains avariable a and β chains (also known as TCRa and TCRp, respectively) or avariable γ and δ chains (also known as TCRy and TCR5, respectively) andthat is capable of specifically binding to an antigen peptide bound to aMHC receptor. In some embodiments, the TCR is in the αβ form. Typically,TCRs that exist in αβ and γδ forms are generally structurally similar,but T cells expressing them may have distinct anatomical locations orfunctions. A TCR can be found on the surface of a cell or in solubleform. Generally, a TCR is found on the surface of T cells (or Tlymphocytes) where it is generally responsible for recognizing antigensbound to major histocompatibility complex (MHC) molecules. In someembodiments, a TCR also can contain a constant domain, a transmembranedomain and/or a short cytoplasmic tail (see, e.g., Janeway et ah,Immunobiology: The Immune System in Health and Disease, 3 rd Ed.,Current Biology Publications, p. 4:33, 1997). For example, in someaspects, each chain of the TCR can possess one N-terminal immunoglobulinvariable domain, one immunoglobulin constant domain, a transmembraneregion, and a short cytoplasmic tail at the C-terminal end. In someembodiments, a TCR is associated with invariant proteins of the CD3complex involved in mediating signal transduction. Unless otherwisestated, the term “TCR” should be understood to encompass functional TCRfragments thereof. The term also encompasses intact or full-length TCRs,including TCRs in the αβ form or γδ form.

Thus, for purposes herein, reference to a TCR includes any TCR orfunctional fragment, such as an antigen-binding portion of a TCR thatbinds to a specific antigenic peptide bound in an MHC molecule, i.e.MHC-peptide complex. An “antigen-binding portion” or antigen-bindingfragment” of a TCR, which can be used interchangeably, refers to amolecule that contains a portion of the structural domains of a TCR, butthat binds the antigen (e.g. MHC-peptide complex) to which the full TCRbinds. In some cases, an antigen-binding portion contains the variabledomains of a TCR, such as variable a chain and variable β chain of aTCR, sufficient to form a binding site for binding to a specificMHC-peptide complex, such as generally where each chain contains threecomplementarity determining regions.

In some embodiments, the variable domains of the TCR chains associate toform loops, or complementarity determining regions (CDRs) analogous toimmunoglobulins, which confer antigen recognition and determine peptidespecificity by forming the binding site of the TCR molecule anddetermine peptide specificity. Typically, like immunoglobulins, the CDRsare separated by framework regions (FRs) {see, e.g., Jores et al., Pwc.Nat'l Acad. Sci. U.S.A. 87:9138, 1990; Chothia et al., EMBO J. 7:3745,1988; see also Lefranc et al., Dev. Comp. Immunol. 27:55, 2003). In someembodiments, CDR3 is the main CDR responsible for recognizing processedantigen, although CDR1 of the alpha chain has also been shown tointeract with the N-terminal part of the antigenic peptide, whereas CDR1of the beta chain interacts with the C-terminal part of the peptide.CDR2 is thought to recognize the MHC molecule. In some embodiments, thevariable region of the β-chain can contain a further hypervariability(HV4) region.

In some embodiments, the TCR chains contain a constant domain. Forexample, like immunoglobulins, the extracellular portion of TCR chains{e.g., a-chain, β-chain) can contain two immunoglobulin domains, avariable domain {e.g., Va or Vp; typically amino acids 1 to 116 based onKabat numbering Kabat et al., “Sequences of Proteins of ImmunologicalInterest, US Dept. Health and Human Services, Public Health ServiceNational Institutes of Health, 1991, 5th ed.) at the N-terminus, and oneconstant domain {e.g., a-chain constant domain or Ca, typically aminoacids 117 to 259 based on Kabat, β-chain constant domain or Cp,typically amino acids 117 to 295 based on Kabat) adjacent to the cellmembrane. For example, in some cases, the extracellular portion of theTCR formed by the two chains contains two membrane-proximal constantdomains, and two membrane-distal variable domains containing CDRs. Theconstant domain of the TCR domain contains short connecting sequences inwhich a cysteine residue forms a disulfide bond, making a link betweenthe two chains. In some embodiments, a TCR may have an additionalcysteine residue in each of the a and β chains such that the TCRcontains two disulfide bonds in the constant domains.

In some embodiments, the TCR chains can contain a transmembrane domain.In some embodiments, the transmembrane domain is positively charged. Insome cases, the TCR chains contain a cytoplasmic tail. In some cases,the structure allows the TCR to associate with other molecules like CD3.For example, a TCR containing constant domains with a transmembraneregion can anchor the protein in the cell membrane and associate withinvariant subunits of the CD3 signaling apparatus or complex.

Generally, CD3 is a multi-protein complex that can possess threedistinct chains (γ, δ, and ε) in mammals and the ζ-chain. For example,in mammals the complex can contain a CD3y chain, a CD35 chain, two CD3schains, and a homodimer of CD3ζ chains. The CD3y, CD35, and CD3s chainsare highly related cell surface proteins of the immunoglobulinsuperfamily containing a single immunoglobulin domain. The transmembraneregions of the CD3y, CD35, and CD3s chains are negatively charged, whichis a characteristic that allows these chains to associate with thepositively charged T cell receptor chains. The intracellular tails ofthe CD3y, CD35, and CD3s chains each contain a single conserved motifknown as an immunoreceptor tyrosine-based activation motif or ITAM,whereas each CD3ζ chain has three. Generally, ITAMs are involved in thesignaling capacity of the TCR complex. These accessory molecules havenegatively charged transmembrane regions and play a role in propagatingthe signal from the TCR into the cell. The CD3- and ζ-chains, togetherwith the TCR, form what is known as the T cell receptor complex.

In some embodiments, the TCR may be a heterodimer of two chains a and β(or optionally γ and δ) or it may be a single chain TCR construct. Insome embodiments, the TCR is a heterodimer containing two separatechains (a and β chains or γ and δ chains) that are linked, such as by adisulfide bond or disulfide bonds.

Exemplary antigen receptors, including CARs and recombinant TCRs, aswell as methods for engineering and introducing the receptors intocells, include those described, for example, in international patentapplication publication numbers WO200014257, WO2013126726,WO2012/129514, WO2014031687, WO2013/166321, WO2013/071154, WO2013/123061U.S. patent application publication numbers US2002131960, US2013287748,US20130149337, U.S. Pat. Nos. 6,451,995, 7,446,190, 8,252,592,8,339,645, 8,398,282, 7,446,179, 6,410,319, 7,070,995, 7,265,209,7,354,762, 7,446,191, 8,324,353, and 8,479,118, and European patentapplication number EP2537416, and/or those described by Sadelain et al.,Cancer Discov. 2013 April; 3(4): 388-398; Davila et al. (2013) PLoS ONE8(4): e61338; Turtle et al., Curr. Opin. Immunol., 2012 October; 24(5):633-39; Wu et al., Cancer, 2012 Mar. 18(2): 160-75. In some aspects, thegenetically engineered antigen receptors include a CAR as described inU.S. Pat. No. 7,446,190, and those described in International PatentApplication Publication No.: WO/2014055668 AI.

Antigens

Among the antigens targeted by the genetically engineered antigenreceptors are those expressed in the context of a disease, condition, orcell type to be targeted via the adoptive cell therapy. Among thediseases and conditions are proliferative, neoplastic, and malignantdiseases and disorders, more particularly cancers.

The cancer may be a solid cancer or a “liquid tumor” such as cancersaffecting the blood, bone marrow and lymphoid system, also known astumors of the hematopoietic and lymphoid tissues, which notably includeleukemia and lymphoma. Liquid tumors include for example acutemyelogenous leukemia (AML), chronic myelogenous leukemia (CML), acutelymphocytic leukemia (ALL), and chronic lymphocytic leukemia (CLL),(including various lymphomas such as mantle cell lymphoma, non-Hodgkinslymphoma (NHL), adenoma, squamous cell carcinoma, laryngeal carcinoma,gallbladder and bile duct cancers, cancers of the retina such asretinoblastoma).

Solid cancers notably include cancers affecting one of the organsselected from the group consisting of colon, rectum, skin, endometrium,lung (including non-small cell lung carcinoma), uterus, bones (such asOsteosarcoma, Chondrosarcomas, Ewing's sarcoma, Fibrosarcomas, Giantcell tumors, Adamantinomas, and Chordomas), liver, kidney, esophagus,stomach, bladder, pancreas, cervix, brain (such as Meningiomas,Glioblastomas, Lower-Grade Astrocytomas, Oligodendrocytomas, PituitaryTumors, Schwannomas, and Metastatic brain cancers), ovary, breast, headand neck region, testis, prostate and the thyroid gland.

Preferably, a cancer according to the invention is a cancer affectingthe blood, bone marrow and lymphoid system as described above.Typically, the cancer is, or is associated, with multiple myeloma.

Diseases according to the invention also encompass infectious diseasesor conditions, such as, but not limited to, viral, retroviral,bacterial, and protozoal infections, immunodeficiency, Cytomegalovirus(CMV), Epstein-Barr virus (EBV), adenovirus, BK polyomavirus; autoimmuneor inflammatory diseases or conditions, such as arthritis, e.g.,rheumatoid arthritis (RA), Type I diabetes, systemic lupus erythematosus(SLE), inflammatory bowel disease, psoriasis, scleroderma, autoimmunethyroid disease, Grave's disease, Crohn's disease multiple sclerosis,asthma, and/or diseases or conditions associated with transplant.

In some embodiments, the antigen is a polypeptide. In some embodiments,it is a carbohydrate or other molecule. In some embodiments, the antigenis selectively expressed or overexpressed on cells of the disease orcondition, e.g., the tumor or pathogenic cells, as compared to normal ornon-targeted cells or tissues. In other embodiments, the antigen isexpressed on normal cells and/or is expressed on the engineered cells.In some such embodiments, a multi-targeting and/or gene disruptionapproach as provided herein is used to improve specificity and/orefficacy.

In some embodiments, the antigen is a universal tumor antigen. The term“universal tumor antigen” refers to an immunogenic molecule, such as aprotein, that is, generally, expressed at a higher level in tumor cellsthan in non-tumor cells and also is expressed in tumors of differentorigins. In some embodiments, the universal tumor antigen is expressedin more than 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90% or more ofhuman cancers. In some embodiments, the universal tumor antigen isexpressed in at least three, at least four, at least five, at least six,at least seven, at least eight or more different types of tumors. Insome cases, the universal tumor antigen may be expressed in non-tumorcells, such as normal cells, but at lower levels than it is expressed intumor cells. In some cases, the universal tumor antigen is not expressedat all in non-tumor cells, such as not expressed in normal cells.Exemplary universal tumor antigens include, for example, humantelomerase reverse transcriptase (hTERT), survivin, mouse double minute2 homolog (MDM2), cytochrome P450 1B1 (CYP1B), HER2/neu, Wilms' tumorgene 1 (WT1), livin, alphafetoprotein (AFP), carcinoembryonic antigen(CEA), mucin 16 (MUC16), MUC1, prostate-specific membrane antigen(PSMA), p53 or cyclin (DI). Peptide epitopes of tumor antigens,including universal tumor antigens, are known in the art and, in someaspects, can be used to generate MHC-restricted antigen receptors, suchas TCRs or TCR-like CARs (see e.g. published PCT application No.WO2011009173 or WO2012135854 and published U.S. application No.US20140065708).

In some aspects, the antigen is expressed on multiple myeloma, such asCD38, CD138, and/or CS-1. Other exemplary multiple myeloma antigensinclude CD56, TIM-3, CD33, CD 123, and/or CD44. Antibodies orantigen-binding fragments directed against such antigens are known andinclude, for example, those described in U.S. Pat. Nos. 8,153,765;8,603,477, 8,008,450; U.S. published application No. US20120189622; andpublished international PCT application Nos. WO2006099875, WO2009080829or WO2012092612. In some embodiments, such antibodies or antigen-bindingfragments thereof (e.g. scFv) can be used to generate a CAR.

In some embodiments, the antigen may be one that is expressed orupregulated on cancer or tumor cells, but that also may be expressed inan immune cell, such as a resting or activated T cell. For example, insome cases, expression of hTERT, survivin and other universal tumorantigens are reported to be present in lymphocytes, including activatedT lymphocytes (see e.g., Weng et al. (1996) J Exp. Med., 183:2471-2479;Hathcock et al. (1998) J Immunol, 160:5702-5706; Liu et al. (1999) Proc.Natl Acad Sci., 96:5147-5152; Turksma et al. (2013) Journal ofTranslational Medicine, 11: 152). Likewise, in some cases, CD38 andother tumor antigens also can be expressed in immune cells, such as Tcells, such as upregulated in activated T cells. For example, in someaspects, CD38 is a known T cell activation marker.

In some embodiments as provided herein, an immune cell, such as a Tcell, can be engineered to repress or disrupt the gene encoding theantigen in the immune cell so that the expressed genetically engineeredantigen receptor does not specifically bind the antigen in the contextof its expression on the immune cell itself. Thus, in some aspects, thismay avoid off-target effects, such as binding of the engineered immunecells to themselves, which may reduce the efficacy of the engineered inthe immune cells, for example, in connection with adoptive cell therapy.

In some embodiments, such as in the case of an inhibitory CAR, thetarget is an off-target marker, such as an antigen not expressed on thediseased cell or cell to be targeted, but that is expressed on a normalor non-diseased cell which also expresses a disease-specific targetbeing targeted by an activating or stimulatory receptor in the sameengineered cell. Exemplary such antigens are MHC molecules, such as MHCclass I molecules, for example, in connection with treating diseases orconditions in which such molecules become downregulated but remainexpressed in non-targeted cells.

In some embodiments, the engineered immune cells can contain an antigenthat targets one or more other antigens. In some embodiments, the one ormore other antigens is a tumor antigen or cancer marker. Other antigentargeted by antigen receptors on the provided immune cells can, in someembodiments, include orphan tyrosine kinase receptor ROR1, tEGFR, Her2,LI-CAM, CD19, CD20, CD22, mesothelin, CEA, and hepatitis B surfaceantigen, anti-folate receptor, CD23, CD24, CD30, CD33, CD38, CD44, EGFR,EGP-2, EGP-4, EPHa2, ErbB2, 3, or 4, FBP, fetal acethycholine ereceptor, GD2, GD3, HMW-MAA, IL-22R-alpha, IL-13R-alpha2, kdr, kappalight chain, Lewis Y, LI-cell adhesion molecule, MAGE-A1, mesothelin,MUC1, MUC16, PSCA, NKG2D Ligands, NY-ESO-1, MART-1, gplOO, oncofetalantigen, ROR1, TAG72, VEGF-R2, carcinoembryonic antigen (CEA), prostatespecific antigen, PSMA, Her2/neu, estrogen receptor, progesteronereceptor, ephrinB2, CD 123, CS-1, c-Met, GD-2, and MAGE A3, CE7, WilmsTumor 1 (WT-1), a cyclin, such as cyclin AI (CCNA1), and/or biotinylatedmolecules, and/or molecules expressed by HIV, HCV, HBV or otherpathogens.

In some embodiments, the CAR binds a pathogen-specific antigen. In someembodiments, the CAR is specific for viral antigens (such as HIV, HCV,HBV, etc.), bacterial antigens, and/or parasitic antigens.

In some embodiments, the cells of the invention is geneticallyengineered to express two or more genetically engineered receptors onthe cell, each recognizing a different antigen and typically eachincluding a different intracellular signaling component. Suchmulti-targeting strategies are described, for example, in InternationalPatent Application, Publication No.: WO 2014055668 AI (describingcombinations of activating and costimulatory CARs, e.g., targeting twodifferent antigens present individually on off-target, e.g., normalcells, but present together only on cells of the disease or condition tobe treated) and Fedorov et al., Sci. Transl. Medicine, 5(215) (December,2013) (describing cells expressing an activating and an inhibitory CAR,such as those in which the activating CAR binds to one antigen expressedon both normal or non-diseased cells and cells of the disease orcondition to be treated, and the inhibitory CAR binds to another antigenexpressed only on the normal cells or cells which it is not desired totreat).

In some contexts, overexpression of a stimulatory factor (for example, alymphokine or a cytokine) may be toxic to a subject. Thus, in somecontexts, the engineered cells include gene segments that cause thecells to be susceptible to negative selection in vivo, such as uponadministration in adoptive immunotherapy. For example in some aspects,the cells are engineered so that they can be eliminated as a result of achange in the in vivo condition of the patient to which they areadministered. The negative selectable phenotype may result from theinsertion of a gene that confers sensitivity to an administered agent,for example, a compound. Negative selectable genes include the Herpessimplex virus type I thymidine kinase (HSV-I TK) gene (Wigler et al.,Cell II:223, 1977) which confers ganciclovir sensitivity; the cellularhypoxanthine phosphribosyltransferase (HPRT)gene, the cellular adeninephosphoribosyltransferase (APRT) gene, bacterial cytosine deaminase,(Mullen et al., Proc. Natl. Acad. Sci. USA. 89:33 (1992)).

In other embodiments of the invention, the cells, e.g., T cells, are notengineered to express recombinant receptors, but rather includenaturally occurring antigen receptors specific for desired antigens,such as tumor-infiltrating lymphocytes and/or T cells cultured in vitroor ex vivo, e.g., during the incubation step(s), to promote expansion ofcells having particular antigen specificity. For example, in someembodiments, the cells are produced for adoptive cell therapy byisolation of tumor-specific T cells, e.g. autologous tumor infiltratinglymphocytes (TIL). The direct targeting of human tumors using autologoustumor infiltrating lymphocytes can in some cases mediate tumorregression (see Rosenberg S A, et al. (1988) N Engl J Med. 319:1676-1680). In some embodiments, lymphocytes are extracted from resectedtumors. In some embodiments, such lymphocytes are expanded in vitro. Insome embodiments, such lymphocytes are cultured with lymphokines (e.g.,IL-2). In some embodiments, such lymphocytes mediate specific lysis ofautologous tumor cells but not allogeneic tumor or autologous normalcells.

Among additional nucleic acids, e.g., genes for introduction are thoseto improve the efficacy of therapy, such as by promoting viabilityand/or function of transferred cells; genes to provide a genetic markerfor selection and/or evaluation of the cells, such as to assess in vivosurvival or localization; genes to improve safety, for example, bymaking the cell susceptible to negative selection in vivo as describedby Lupton S. D. et al., Mol. and Cell Biol., 11:6 (1991); and Riddell etal., Human Gene Therapy 3:319-338 (1992); see also the publications ofPCT/US91/08442 and PCT/US94/05601 by Lupton et al. describing the use ofbifunctional selectable fusion genes derived from fusing a dominantpositive selectable marker with a negative selectable marker. See, e.g.,Riddell et al., U.S. Pat. No. 6,040,177, at columns 14-17.

Method for Obtaining Cells According to the Invention

The present invention also relates to a method of producing a modifiedor engineered immune cell, comprising a step consisting in inhibiting ofthe expression and/or activity of Suv39h1 in the immune cell.

Preferably, the method for obtaining cells according to the inventionfurther comprises a step consisting in introducing into said immunecells of a genetically engineered antigen receptor that specificallybinds to a target antigen.

The inhibition of the expression and/or activity of Suv39h1 and theintroduction of a genetically engineered antigen receptor thatspecifically binds to a target antigen in the immune cell can be carriedout simultaneously or sequentially in any order.

Inhibition of Suv39h1

According to the invention, the engineered immune cell can be contactedwith at least one agent that inhibits or blocks the expression and/oractivity of Suv39h1.

Said agent can be selected from small molecule inhibitors; antibodiesderivatives such as intrabodies, nanobodies or affibodies that typicallyblock or inhibit Suv39h1 expression or activity; aptamers that typicallyblock or inhibit Suv39h1 expression or activity; nucleic acid moleculesthat block transcription or translation, such as antisense moleculescomplementary to Suv39h1; RNA interfering agents (such as a smallinterfering RNA (siRNA), small hairpin RNA (shRNA), microRNA (miRNA), ora piwiRNA (piRNA); ribozymes an combination thereof.

The at least one agent can also be an exogenous nucleic acid comprisinga) an engineered, non-naturally occurring Clustered RegularlyInterspaced Short Palindromic Repeats (CRISPR) guide RNA that hybridizeswith Suv39h1 genomic nucleic acid sequence and/or b) a nucleotidesequence encoding a CRISPR protein (typically a Type-II Cas9 protein),optionally wherein the cells are transgenic for expressing a Cas9protein. The agent may also be a Zinc finger protein (ZFN) or a TALprotein.

The term “small organic molecule” refers to a molecule of a sizecomparable to those organic molecules generally used in pharmaceuticals.The term excludes biological macro molecules (e. g., proteins, nucleicacids, etc.). Preferred small organic molecules range in size up toabout 5000 Da, more preferably up to 2000 Da, and most preferably up toabout 1000 Da.

In a particular embodiment, the inhibitor of H3K9-histonemethyltransferase SUV39H1 is chaetocin (CAS 28097-03-2) as described byGreiner D, Bonaldi T, Eskeland R, Roemer E, Imhof A. “Identification ofa specific inhibitor of the histone methyltransferase SU(VAR)3-9”. NatChem Biol. 2005 August; l(3): 143-5.; Weber, H. P., et al, “Themolecular structure and absolute configuration of chaetocin”, ActaCryst, B28, 2945-2951 (1972); Udagawa, S., et al, “The production ofchaetoglobosins, sterigmatocystin, O-methylsterigmatocystin, andchaetocin by Chaetomium spp. and related fungi”, Can. J. microbiol, 25,170-177 (1979); and Gardiner, D. M., et al, “Theepipolythiodioxopiperazine (ETP) class of fungal toxins: distribution,mode of action, functions and biosynthesis”, Microbiol, 151, 1021-1032(2005). For example, chaetocin is commercially available from SigmaAldrich.

An inhibitor of Suv39h1 can also be ETP69(Rac-(3S,6S,7S,8a5)-6-(benzo[d][1,3]dioxol-5-yl)-2,3,7-trimethyl-1,4-dioxohexahydro-6H-3,8a-epidithiopyrrolo[1,2-a]pyrazine-7-carbonitrile),a racemic analog of the epidithiodiketopiperazine alkaloid chaetocin A(see WO2014066435 but see also Baumann M, Dieskau A P, Loertscher B M,et al. Tricyclic Analogues of Epidithiodioxopiperazine Alkaloids withPromising In Vitro and In Vivo Antitumor Activity. Chemical science(Royal Society of Chemistry: 2010). 2015; 6:4451-4457, and Snigdha S,Prieto G A, Petrosyan A, et al. H3K9me3 Inhibition Improves Memory,Promotes Spine Formation, and Increases BDNF Levels in the AgedHippocampus. The Journal of Neuroscience. 2016; 36(12):3611-3622).

The inhibiting activity of a compound may be determined using variousmethods as described in Greiner D. Et al. Nat Chem Biol. 2005 August;l(3): 143-5 or Eskeland, R. et al. Biochemistry 43, 3740-3749 (2004).

Inhibition of Suv39h1 in the cell can be achieved before or afterinjection in the targeted patient. In some embodiment, inhibition aspreviously defined is performed in vivo after administration of the cellto the subject. Typically a Suv39h1 inhibitor as herein defined can beincluded in the composition containing the cell. Suv39h1 may also beadministered separately before, concomitantly of after administration ofthe cell(s) to the subject.

Typically, inhibition of Suv39h1 according to the invention may beachieved with incubation of a cell according to the invention with acomposition containing at least one pharmacological inhibitor aspreviously described. The inhibitor is included during the expansion ofthe anti-tumor T cells in vitro, thus modifying their reconstitution,survival and therapeutic efficacy after adoptive transfer.

Inhibition of Suv39h1 in a cell according to the invention may beachieved with intrabodies. Intrabodies are antibodies that bindintracellularly to their antigen after being produced in the same cell(for a review se for example, Marschall A L, Dübel S and Böldicke T“Specific in vivo knockdown of protein function by intrabodies”, MAbs.2015; 7(6):1010-35. but see also Van Impe K, Bethuyne J, Cool S, ImpensF, Ruano-Gallego D, De Wever O, Vanloo B, Van Troys M, Lambein K,Boucherie C, et al. “A nanobody targeting the F-actin capping proteinCapG restrains breast cancer metastasis”. Breast Cancer Res 2013;15:R116; Hyland S, Beerli R R, Barbas C F, Hynes N E, Wels W.“Generation and functional characterization of intracellular antibodiesinteracting with the kinase domain of human EGF receptor. Oncogene 2003;22:1557-67”; Lobato M N, Rabbitts T H. “Intracellular antibodies andchallenges facing their use as therapeutic agents”. Trends Mol Med 2003;9:390-6, and Donini M, Morea V, Desiderio A, Pashkoulov D, Villani M E,Tramontano A, Benvenuto E. “Engineering stable cytoplasmic intrabodieswith designed specificity”. J Mol Biol. 2003 Jul. 4; 330(2):323-32.).

Intrabodies can be generated by cloning the respective cDNA from anexisting hybridoma clone or more conveniently, new scFvs/Fabs can beselected from in vitro display techniques such as phage display whichprovide the necessary gene encoding the antibody from the onset andallow a more detailed predesign of antibody fine specificity. Inaddition, bacterial-, yeast-, mammalian cell surface display andribosome display can be employed. However, the most commonly used invitro display system for selection of specific antibodies is phagedisplay. In a procedure called panning (affinity selection), recombinantantibody phages are selected by incubation of the antibody phagerepertoire with the antigen. This process is repeated several timesleading to enriched antibody repertoires comprising specific antigenbinders to almost any possible target. To date, in vitro assembledrecombinant human antibody libraries have already yielded thousands ofnovel recombinant antibody fragments. It is to be noted that theprerequisite for a specific protein knockdown by a cytoplasmic intrabodyis that the antigen is neutralized/inactivated through the antibodybinding. Five different approaches to generate suitable antibodies haveemerged: 1) In vivo selection of functional intrabodies in eukaryotessuch as yeast and in prokaryotes such as E. coli (antigen-dependent andindependent); 2) generation of antibody fusion proteins for improvingcytosolic stability; 3) use of special frameworks for improvingcytosolic stability (e.g., by grafting CDRs or introduction of syntheticCDRs in stable antibody frameworks); 4) use of single domain antibodiesfor improved cytosolic stability; and 5) selection of disulfide bondfree stable intrabodies. Those approaches are notably detailed inMarschall, A. L et al., mAbs 2015 as mentioned above.

The most commonly used format for intrabodies is the scFv, whichconsists of the H- and L-chain variable antibody domain (VH and VL) heldtogether by a short, flexible linker sequence (frequently (Gly4Ser)3),to avoid the need for separate expression and assembly of the 2 antibodychains of a full IgG or Fab molecule. Alternatively, the Fab formatcomprising additionally the C1 domain of the heavy chain and theconstant region of the light chain has been used. Recently, a newpossible format for intrabodies, the scFab, has been described. ThescFab format promises easier subcloning of available Fab genes into theintracellular expression vector, but it remains to be seen whether thisprovides any advantage over the well-established scFv format. Inaddition to scFv and Fab, bispecific formats have been used asintrabodies. A bispecific Tie-2×VEGFR-2 antibody targeted to the ERdemonstrated an extended half-life compared to the monospecific antibodycounterparts. A bispecific transmembrane intrabody has been developed asa special format to simultaneously recognize intra- and extracellularepitopes of the epidermal growth factor, combining the distinct featuresof the related monospecific antibodies, i.e., inhibition ofautophosphorylation and ligand binding.

Another intrabody format particularly suitable for cytoplasmicexpression are single domain antibodies (also called nanobodies) derivedfrom camels or consisting of one human VH domain or human VL domain.These single domain antibodies often have advantageous properties, e.g.,high stability; good solubility; ease of library cloning and selection;high expression yield in E. coli and yeast.

The intrabody gene can be expressed inside the target cell aftertransfection with an expression plasmid or viral transduction with arecombinant virus. Typically, the choice is aimed at providing optimalintrabody transfection and production levels. Successful transfectionand subsequent intrabody production can be analyzed by immunoblotdetection of the produced antibody, but, for the evaluation of correctintrabody/antigen-interaction, co-immunoprecipitation from HEK 293 cellextracts transiently cotransfected with the corresponding antigen andintrabody expression plasmids may be used.

Inhibition of Suv39h1 in a cell according to the invention may also beeffected with aptamers that inhibit or block Suv39h1 expression oractivity. Aptamers are a class of molecule that represents analternative to antibodies in term of molecular recognition. Aptamers areoligonucleotide (DNA or RNA) or oligopeptide sequences with the capacityto recognize virtually any class of target molecules with high affinityand specificity.

Oligonucleotide aptamers may be isolated through Systematic Evolution ofLigands by Exponential enrichment (SELEX) of a random sequence library,as described in Tuerk C. and Gold L., 1990. The random sequence libraryis obtainable by combinatorial chemical synthesis of DNA. In thislibrary, each member is a linear oligomer, eventually chemicallymodified, of a unique sequence. Possible modifications, uses andadvantages of this class of molecules have been reviewed in Jayasena S.D., 1999.

Peptide aptamers consists of a conformationally constrained antibodyvariable region displayed by a platform protein, such as E. coliThioredoxin A that are selected from combinatorial libraries by twohybrid methods (Colas P, Cohen B, Jessen T, Grishina I, McCoy J, BrentR. “Genetic selection of peptide aptamers that recognize and inhibitcyclin-dependent kinase 2”. Nature. 1996 Apr. 11; 380(6574):548-50).

Inhibition of Suv39h1 in a cell according to the invention may also beeffected with affibody molecules. Affibody are small proteins engineeredto bind to a large number of target proteins or peptides with highaffinity, imitating monoclonal antibodies, and are therefore a member ofthe family of antibody mimetics (see for review Lofblom J, Feldwisch J,Tolmachev V, Carlsson J, Ståhl S, Frejd F Y. Affibody molecules:engineered proteins for therapeutic, diagnostic and biotechnologicalapplications. FEBS Lett. 2010 Jun. 18; 584(12):2670-80). Affibodymolecules are based on an engineered variant (the Z domain) of theB-domain in the immunoglobulin-binding regions of staphylococcal proteinA, with specific binding for theoretically any given target. Affibodymolecule libraries are generally constructed by combinatorialrandomization of 13 amino acid positions in helices one and two thatcomprise the original Fc-binding surface of the Z-domain. The librarieshave typically been displayed on phages, followed by biopanning againstdesired targets. Should the affinity of the primary be increased,affinity maturation generally results in improved binders and may beachieved by either helix shuffling or sequence alignment combined withdirected combinatorial mutagenesis. The newly identified molecules withtheir altered binding surface generally keep the original helicalstructure as well as the high stability, although unique exceptions withinteresting properties have been reported. Due to their small size andrapid folding properties, affibody molecules can be produced by chemicalpeptide synthesis.

In other embodiments of the invention, inhibition of Suv39h1 activitycan be achieved by gene repression/suppression via gene knockdown usingRNA interference (RNAi) such as short interfering RNA (siRNA) shorthairpin RNA (shRNA) or ribozymes. siRNA technology includes that basedon RNAi utilizing a double-stranded RNA molecule having a sequencehomologous with the nucleotide sequence of mRNA which is transcribedfrom the gene, and a sequence complementary with the nucleotidesequence. siRNA generally is homologous/complementary with one region ofmRNA which is transcribed from the gene, or may be siRNA including aplurality of RNA molecules which are homologous/complementary withdifferent regions.

Anti-sense oligonucleotides, including anti-sense RNA molecules andanti-sense DNA molecules, would act to directly block the translation ofH3K9-histone methyltransferase Suv39h1 and thus preventing proteintranslation or increasing mRNA degradation, thus decreasing the level ofH3K9-histone methyltransferase SUV39H1 and thus its activity in a cell.For example, antisense oligonucleotides of at least about 15 bases andcomplementary to unique regions of the mRNA transcript sequence encodingH3K9-histone methyltransferase SUV39H1 can be synthesized, e.g., byconventional phosphodiester techniques and administered by e.g.,intravenous injection or infusion. Methods for using antisensetechniques for specifically inhibiting gene expression of genes whosesequence is known are well known in the art (see for example U.S. Pat.Nos. 6,566,135; 6,566,131; 6,365,354; 6,410,323; 6,107,091; 6,046,321;and 5,981,732).

An “RNA interfering agent” as used herein, is defined as any agent,which interferes with or inhibits expression of a target biomarker geneby RNA interference (RNAi). Such RNA interfering agents include, but arenot limited to, nucleic acid molecules including RNA molecules, whichare homologous to the target gene of the invention (e.g., Suv39h1), or afragment thereof, short interfering RNA (siRNA), and small moleculeswhich interfere with or inhibit expression of the target nucleic acid byRNA interference (RNAi).

Small inhibitory RNAs (siRNAs) can also function as inhibitors ofexpression for use in the present invention. H3K9-histonemethyltransferase SUV39H1 gene expression can be reduced by contacting asubject or cell with a small double stranded RNA (dsRNA), or a vector orconstruct causing the production of a small double stranded RNA, suchthat H3K9-histone methyltransferase SUV39H1 gene expression isspecifically inhibited (i.e. RNA interference or RNAi). Methods forselecting an appropriate dsRNA or dsRNA-encoding vector are well knownin the art for genes whose sequence is known (see for example Tuschl, T.et al. (1999); Elbashir, S. M. et al. (2001); Hannon, G J. (2002);McManus, M T. et al. (2002); Brummelkamp, T R. et al. (2002); U.S. Pat.Nos. 6,573,099 and 6,506,559; and International Patent Publication Nos.WO 01/36646, WO 99/32619, and WO 01/68836). All or part of thephosphodiester bonds of the siRNAs of the invention are advantageouslyprotected. This protection is generally implemented via the chemicalroute using methods that are known by art. The phosphodiester bonds canbe protected, for example, by a thiol or amine functional group or by aphenyl group. The 5′- and/or 3′-ends of the siRNAs of the invention arealso advantageously protected, for example, using the techniquedescribed above for protecting the phosphodiester bonds. The siRNAssequences advantageously comprise at least twelve contiguousdinucleotides or their derivatives.

As used herein, the term “siRNA derivatives” with respect to the presentnucleic acid sequences refers to a nucleic acid having a percentage ofidentity of at least 90% with erythropoietin or fragment thereof,preferably of at least 95%, as an example of at least 98%, and morepreferably of at least 98%.

As used herein, the expression “percentage of identity” between twonucleic acid sequences, means the percentage of identical nucleic acid,between the two sequences to be compared, obtained with the bestalignment of said sequences, this percentage being purely statisticaland the differences between these two sequences being randomly spreadover the nucleic acid acids sequences. As used herein, “best alignment”or “optimal alignment”, means the alignment for which the determinedpercentage of identity (see below) is the highest. Sequence comparisonbetween two nucleic acids sequences is usually realized by comparingthese sequences that have been previously aligned according to the bestalignment; this comparison is realized on segments of comparison inorder to identify and compared the local regions of similarity.

The best sequences alignment to perform comparison can be realized,besides manually, by using the global homology algorithm developed bySMITH and WATERMAN (Ad. App. Math., vol. 2, p:482, 1981), by using thelocal homology algorithm developed by NEDDLEMAN and WUNSCH (J. Mol.Biol, vol. 48, p:443, 1970), by using the method of similaritiesdeveloped by PEARSON and LIPMAN (Proc. Natl. Acd. Sci. USA, vol. 85,p:2444, 1988), by using computer softwares using such algorithms (GAP,BESTFIT, BLAST P, BLAST N, FASTA, TFASTA in the Wisconsin Geneticssoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis. USA), by using the MUSCLE multiple alignment algorithms (Edgar,Robert C, Nucleic Acids Research, vol. 32, p: 1792, 2004). To get thebest local alignment, one can preferably use BLAST software. Theidentity percentage between two sequences of nucleic acids is determinedby comparing these two sequences optimally aligned, the nucleic acidssequences being able to comprise additions or deletions in respect tothe reference sequence in order to get the optimal alignment betweenthese two sequences. The percentage of identity is calculated bydetermining the number of identical positions between these twosequences, and dividing this number by the total number of comparedpositions, and by multiplying the result obtained by 100 to get thepercentage of identity between these two sequences.

shRNAs (short hairpin RNA) can also function as inhibitors of expressionfor use in the present invention. shRNAs are typically composed of ashort (e.g., 19-25 nucleotide) antisense strand, followed by a 5-9nucleotide loop, and the analogous sense strand. Alternatively, thesense strand may precede the nucleotide loop structure and the antisensestrand may follow.

Ribozymes can also function as inhibitors of expression for use in thepresent invention. Ribozymes are enzymatic RNA molecules capable ofcatalyzing the specific cleavage of RNA. The mechanism of ribozymeaction involves sequence specific hybridization of the ribozyme moleculeto complementary target RNA, followed by endonucleo lytic cleavage.Engineered hairpin or hammerhead motif ribozyme molecules thatspecifically and efficiently catalyze endonucleolytic cleavage ofH3K9-histone methyltransferase SUV39H1 mRNA sequences are thereby usefulwithin the scope of the present invention. Specific ribozyme cleavagesites within any potential RNA target are initially identified byscanning the target molecule for ribozyme cleavage sites, whichtypically include the following sequences, GUA, GUU, and GUC. Onceidentified, short RNA sequences of between about 15 and 20ribonucleotides corresponding to the region of the target genecontaining the cleavage site can be evaluated for predicted structuralfeatures, such as secondary structure, that can render theoligonucleotide sequence unsuitable.

Both antisense oligonucleotides and ribozymes useful as inhibitors ofexpression can be prepared by known methods. These include techniquesfor chemical synthesis such as, e.g., by solid phase phosphoramaditechemical synthesis. Alternatively, anti-sense RNA molecules can begenerated by in vitro or in vivo transcription of DNA sequences encodingthe RNA molecule. Such DNA sequences can be incorporated into a widevariety of vectors that incorporate suitable RNA polymerase promoterssuch as the T7 or SP6 polymerase promoters. Various modifications to theoligonucleotides of the invention can be introduced as a means ofincreasing intracellular stability and half-life.

Possible modifications include but are not limited to the addition offlanking sequences of ribonucleotides or deoxyribonucleotides to the 5′and/or 3′ ends of the molecule, or the use of phosphorothioate or2′-0-methyl rather than phosphodiesterase linkages within theoligonucleotide backbone.

Antisense oligonucleotides, siRNAs, shRNAs and ribozymes of theinvention may be delivered in vivo alone or in association with avector. In its broadest sense, a “vector” is any vehicle capable offacilitating the transfer of the antisense oligonucleotide, siRNA, shRNAor ribozyme nucleic acid to the cells and preferably cells expressingH3K9-histone methyltransferase SUV39H1. Preferably, the vectortransports the nucleic acid to cells with reduced degradation relativeto the extent of degradation that would result in the absence of thevector. In general, the vectors useful in the invention include, but arenot limited to, plasmids, phagemids, viruses, other vehicles derivedfrom viral or bacterial sources that have been manipulated by theinsertion or incorporation of the antisense oligonucleotide, siRNA,shRNA or ribozyme nucleic acid sequences. Viral vectors are a preferredtype of vector and include, but are not limited to nucleic acidsequences from the following viruses: retrovirus, such as moloney murineleukemia virus, harvey murine sarcoma virus, murine mammary tumor virus,and rous sarcoma virus; adenovirus, adeno-associated virus; SV40-typeviruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses;herpes virus; vaccinia virus; polio virus; and R A virus such as aretrovirus. One can readily employ other vectors not named but known tothe art.

Preferred viral vectors are based on non-cytopathic eukaryotic virusesin which nonessential genes have been replaced with the gene ofinterest. Non-cytopathic viruses include retroviruses (e.g.,lentivirus), the life cycle of which involves reverse transcription ofgenomic viral RNA into DNA with subsequent proviral integration intohost cellular DNA. Retroviruses have been approved for human genetherapy trials. Most useful are those retroviruses that arereplication-deficient (i.e., capable of directing synthesis of thedesired proteins, but incapable of manufacturing an infectiousparticle). Such genetically altered retroviral expression vectors havegeneral utility for the high-efficiency transduction of genes in vivo.Standard protocols for producing replication-deficient retroviruses(including the steps of incorporation of exogenous genetic material intoa plasmid, transfection of a packaging cell lined with plasmid,production of recombinant retroviruses by the packaging cell line,collection of viral particles from tissue culture media, and infectionof the target cells with viral particles) are provided in Kriegler, 1990and in Murry, 1991).

Preferred viruses for certain applications are the adenoviruses andadeno-associated (AAV) viruses, which are double-stranded DNA virusesthat have already been approved for human use in gene therapy. Actually12 different AAV serotypes (AAV1 to 12) are known, each with differenttissue tropisms (Wu, Z Mol Ther 2006; 14:316-27). Recombinant AAVs arederived from the dependent parvovirus AAV2 (Choi, V W J Virol 2005;79:6801-07). The adeno-associated virus type 1 to 12 can be engineeredto be replication deficient and is capable of infecting a wide range ofcell types and species (Wu, Z Mol Ther 2006; 14:316-27). It further hasadvantages such as, heat and lipid solvent stability; high transductionfrequencies in cells of diverse lineages, including hemopoietic cells;and lack of superinfection inhibition thus allowing multiple series oftransductions. Reportedly, the adeno-associated virus can integrate intohuman cellular DNA in a site-specific manner, thereby minimizing thepossibility of insertional mutagenesis and variability of inserted geneexpression characteristic of retroviral infection. In addition,wild-type adeno-associated virus infections have been followed in tissueculture for greater than 100 passages in the absence of selectivepressure, implying that the adeno-associated virus genomic integrationis a relatively stable event. The adeno-associated virus can alsofunction in an extrachromosomal fashion.

Other vectors include plasmid vectors. Plasmid vectors have beenextensively described in the art and are well known to those of skill inthe art. See e.g. Sambrook et al, 1989. In the last few years, plasmidvectors have been used as DNA vaccines for delivering antigen-encodinggenes to cells in vivo. They are particularly advantageous for thisbecause they do not have the same safety concerns as with many of theviral vectors. These plasmids, however, having a promoter compatiblewith the host cell, can express a peptide from a gene operativelyencoded within the plasmid. Some commonly used plasmids include pBR322,pUC18, pUC19, pRC/CMV, SV40, and pBlueScript. Other plasmids are wellknown to those of ordinary skill in the art. Additionally, plasmids maybe custom designed using restriction enzymes and ligation reactions toremove and add specific fragments of DNA. Plasmids may be delivered by avariety of parenteral, mucosal and topical routes. For example, the DNAplasmid can be injected by intramuscular, intradermal, subcutaneous, orother routes. It may also be administered by intranasal sprays or drops,rectal suppository and orally. It may also be administered into theepidermis or a mucosal surface using a gene-gun. The plasmids may begiven in an aqueous solution, dried onto gold particles or inassociation with another DNA delivery system including but not limitedto liposomes, dendrimers, cochleate delivery vehicles and microencapsulation.

The antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acidsequence according to the invention is generally under the control of aheterologous regulatory region, e.g., a heterologous promoter. Thepromoter may be specific for Muller glial cells, microglia cells,endothelial cells, pericyte cells and astrocytes For example, a specificexpression in Muller glial cells may be obtained through the promoter ofthe glutamine synthetase gene is suitable. The promoter can also be, asa matter of example, a viral promoter, such as CMV promoter or anysynthetic promoters.

Gene Repression or Disruption of Suv39h1

Inhibition of Suv39h1 in a cell according to the invention may also beeffected via repression or disruption of the Suv39h1gene, such as bydeletion, e.g., deletion of the entire gene, exon, or region, and/orreplacement with an exogenous sequence, and/or by mutation, e.g.,frameshift or missense mutation, within the gene, typically within anexon of the gene. In some embodiments, the disruption results in apremature stop codon being incorporated into the gene, such that theSuv39h1 protein is not expressed or is non-functional. The disruption isgenerally carried out at the DNA level. The disruption generally ispermanent, irreversible, or not transient.

In some embodiments, the gene disruption or repression is achieved usinggene editing agents such as a DNA-targeting molecule, such as aDNA-binding protein or DNA-binding nucleic acid, or complex, compound,or composition, containing the same, which specifically binds to orhybridizes to the gene. In some embodiments, the DNA-targeting moleculecomprises a DNA-binding domain, e.g., a zinc finger protein (ZFP)DNA-binding domain, a transcription activator-like protein (TAL) or TALeffector (TALE) DNA-binding domain, a clustered regularly interspacedshort palindromic repeats (CRISPR) DNA-binding domain, or a DNA-bindingdomain from a meganuclease. Zinc finger, TALE, and CRISPR system bindingdomains can be “engineered” to bind to a predetermined nucleotidesequence.

In some embodiments, the DNA-targeting molecule, complex, or combinationcontains a DNA-binding molecule and one or more additional domain, suchas an effector domain to facilitate the repression or disruption of thegene. For example, in some embodiments, the gene disruption is carriedout by fusion proteins that comprise DNA-binding proteins and aheterologous regulatory domain or functional fragment thereof.

Typically, the additional domain is a nuclease domain. Thus, in someembodiments, gene disruption is facilitated by gene or genome editing,using engineered proteins, such as nucleases and nuclease-containingcomplexes or fusion proteins, composed of sequence-specific DNA-bindingdomains fused to, or complexed with, non-specific DNA-cleavage moleculessuch as nucleases.

These targeted chimeric nucleases or nuclease-containing complexes carryout precise genetic modifications by inducing targeted double-strandedbreaks or single-stranded breaks, stimulating the cellular DNA-repairmechanisms, including error-prone nonhomologous end joining (NHEJ) andhomology-directed repair (HDR). In some embodiments the nuclease is anendonuclease, such as a zinc finger nuclease (ZFN), TALE nuclease(TALEN), an RNA-guided endonuclease (RGEN), such as a CRISPR-associated(Cas) protein, or a meganuclease. Such systems are well-known in the art(see, for example, U.S. Pat. No. 8,697,359; Sander and Joung (2014) Nat.Biotech. 32:347-355; Hale et al. (2009) Cell 139:945-956; Karginov andHannon (2010) Mol. Cell 37:7; U.S. Pat. Publ. 2014/0087426 and2012/0178169; Boch et al. (2011) Nat. Biotech. 29: 135-136; Boch et al.(2009) Science 326: 1509-1512; Moscou and Bogdanove (2009) Science 326:1501; Weber et al. (2011) PLoS One 6:e19722; Li et al. (2011) Nucl.Acids Res. 39:6315-6325; Zhang et al. (2011) Nat. Biotech. 29: 149-153;Miller et al. (2011) Nat. Biotech. 29: 143-148; Lin et al. (2014) Nucl.Acids Res. 42:e47). Such genetic strategies can use constitutiveexpression systems or inducible expression systems according towell-known methods in the art.

ZFPs and ZFNs; TALs, TALEs, and TALENs

In some embodiments, the DNA-targeting molecule includes a DNA-bindingprotein such as one or more zinc finger protein (ZFP) or transcriptionactivator-like protein (TAL), fused to an effector protein such as anendonuclease. Examples include ZFNs, TALEs, and TALENs. See Lloyd etal., Frontiers in Immunology, 4(221), 1-7 (2013).

In some embodiments, the DNA-targeting molecule comprises one or morezinc-finger proteins (ZFPs) or domains thereof that bind to DNA in asequence-specific manner. A ZFP or domain thereof is a protein or domainwithin a larger protein, that binds DNA in a sequence-specific mannerthrough one or more zinc fingers regions of amino acid sequence withinthe binding domain whose structure is stabilized through coordination ofa zinc ion. Generally, sequence-specificity of a ZFP may be altered bymaking amino acid substitutions at the four helix positions (-1, 2, 3and 6) on a zinc finger recognition helix. Thus, in some embodiments,the ZFP or ZFP-containing molecule is non-naturally occurring, e.g., isengineered to bind to the target site of choice. See, for example,Beerli et al. (2002) Nature Biotechnol. 20: 135-141; Pabo et al. (2001)Ann. Rev. Biochem. 70:313-340; Isalan et al. (2001) Nature Biotechnol.19:656-660; Segal et al. (2001) Curr. Opin. Biotechnol. 12:632-637; Chooet al. (2000) Curr. Opin. Struct. Biol. 10:411-416.

In some embodiments, the DNA-targeting molecule is or comprises azinc-finger DNA binding domain fused to a DNA cleavage domain to form azinc-finger nuclease (ZFN). In some embodiments, fusion proteinscomprise the cleavage domain (or cleavage half-domain) from at least oneType IIS restriction enzyme and one or more zinc finger binding domains,which may or may not be engineered. In some embodiments, the cleavagedomain is from the Type IIS restriction endonuclease Fok I. See, forexample, U.S. Pat. Nos. 5,356,802; 5,436,150 and 5,487,994; as well asLi et al. (1992) Proc. Natl. Acad. Sci. USA 89:4275-4279; Li et al.(1993) Proc. Natl. Acad. Sci. USA 90:2764-2768; Kim et al. (1994a) Proc.Natl. Acad. Sci. USA 91:883-887; Kim et al. (1994b) J. Biol. Chem.269:31,978-31,982.

In some aspects, the ZFNs efficiently generate a double strand break(DSB), for example at a predetermined site in the coding region of thetargeted gene (i.e. Suv39h1). Typical targeted gene regions includeexons, regions encoding N-terminal regions, first exon, second exon, andpromoter or enhancer regions. In some embodiments, transient expressionof the ZFNs promotes highly efficient and permanent disruption of thetarget gene in the engineered cells. In particular, in some embodiments,delivery of the ZFNs results in the permanent disruption of the genewith efficiencies surpassing 50%. Many gene-specific engineered zincfingers are available commercially. For example, Sangamo Biosciences(Richmond, Calif., USA) has developed a platform (CompoZr) forzinc-finger construction in partnership with Sigma-Aldrich (St. Louis,Mo., USA), allowing investigators to bypass zinc-finger construction andvalidation altogether, and provides specifically targeted zinc fingersfor thousands of proteins. Gaj et al., Trends in Biotechnology, 2013,31(7), 397-405. In some embodiments, commercially available zinc fingersare used or are custom designed. (See, for example, Sigma-Aldrichcatalog numbers CSTZFND, CSTZFN, CTI1-1KT, and PZD0020).

In some embodiments, the DNA-targeting molecule comprises a naturallyoccurring or engineered (non-naturally occurring) transcriptionactivator-like protein (TAL) DNA binding domain, such as in atranscription activator-like protein effector (TALE) protein, See, e.g.,U.S. Patent Publication No. 20110301073. In some embodiments, themolecule is a DNA binding endonuclease, such as a TALE-nuclease (TALEN).In some aspects the TALEN is a fusion protein comprising a DNA-bindingdomain derived from a TALE and a nuclease catalytic domain to cleave anucleic acid target sequence. In some embodiments, the TALE DNA-bindingdomain has been engineered to bind a target sequence within genes thatencode the target antigen and/or the immunosuppressive molecule. Forexample, in some aspects, the TALE DNA-binding domain may target CD38and/or an adenosine receptor, such as A2AR.

In some embodiments, the TALEN recognizes and cleaves the targetsequence in the gene. In some aspects, cleavage of the DNA results indouble-stranded breaks. In some aspects the breaks stimulate the rate ofhomologous recombination or non-homologous end joining (NHEJ).Generally, NHEJ is an imperfect repair process that often results inchanges to the DNA sequence at the site of the cleavage. In someaspects, repair mechanisms involve rejoining of what remains of the twoDNA ends through direct re-ligation (Critchlow and Jackson, TrendsBiochem Sci. 1998 October; 23(10):394-8) or via the so-calledmicrohomology-mediated end joining. In some embodiments, repair via NHEJresults in small insertions or deletions and can be used to disrupt andthereby repress the gene. In some embodiments, the modification may be asubstitution, deletion, or addition of at least one nucleotide. In someaspects, cells in which a cleavage-induced mutagenesis event, i.e. amutagenesis event consecutive to an NHEJ event, has occurred can beidentified and/or selected by well-known methods in the art.

TALE repeats can be assembled to specifically target the Suv39h1 gene.(Gaj et al., Trends in Biotechnology, 2013, 31(7), 397-405). A libraryof TALENs targeting 18,740 human protein-coding genes has beenconstructed (Kim et al., Nature Biotechnology. 31, 251-258 (2013)).Custom-designed TALE arrays are commercially available through CellectisBioresearch (Paris, France), Transposagen Biopharmaceuticals (Lexington,Ky., USA), and Life Technologies (Grand Island, N.Y., USA).Specifically, TALENs that target CD38 are commercially available (SeeGencopoeia, catalog numbers HTN222870-1, HTN222870-2, and HTN222870-3,available on the World Wide Web atwww.genecopoeia.com/product/search/detail.php?prt=26&cid=&key=HTN222870). Exemplary molecules are described, e.g., inU.S. Patent Publication Nos. US 2014/0120622, and 2013/0315884.

In some embodiments the TALENs are introduced as transgenes encoded byone or more plasmid vectors. In some aspects, the plasmid vector cancontain a selection marker which provides for identification and/orselection of cells which received said vector.

RGENs (CRISPR/Cas Systems)

The gene repression can be carried out using one or more DNA-bindingnucleic acids, such as disruption via an RNA-guided endonuclease (RGEN),or other form of repression by another RNA-guided effector molecule. Forexample, in some embodiments, the gene repression can be carried outusing clustered regularly interspaced short palindromic repeats (CRISPR)and CRISPR-associated proteins. See Sander and Joung, NatureBiotechnology, 32(4): 347-355.

In general, “CRISPR system” refers collectively to transcripts and otherelements involved in the expression of, or directing the activity of,CRISPR-associated (“Cas”) genes, including sequences encoding a Casgene, a tracr (trans-activating CRISPR) sequence (e.g. tracrRNA or anactive partial tracrRNA), a tracr-mate sequence (encompassing a “directrepeat” and a tracrRNA-processed partial direct repeat in the context ofan endogenous CRISPR system), a guide sequence (also referred to as a“spacer” in the context of an endogenous CRISPR system), and/or othersequences and transcripts from a CRISPR locus.

Typically, the CRISPR/Cas nuclease or CRISPR/Cas nuclease systemincludes a non-coding RNA molecule (guide) RNA, whichsequence-specifically binds to DNA, and a CRISPR protein, with nucleasefunctionality (e.g., two nuclease domains). One or more elements of aCRISPR system can derive from a type I, type II, or type III CRISPRsystem, such as Cas nuclease. Preferably, the CRISPR protein is a casenzyme such as9. Cas enzymes are well-known in the field; for example,the amino acid sequence of S. pyogenes Cas9 protein may be found in theSwissProt database under accession number Q99ZW2. In some embodiments, aCas nuclease and gRNA are introduced into the cell. In some embodiments,the CRISPR system induces DSBs at the target site, followed bydisruptions as discussed herein. In other embodiments, Cas9 variants,deemed “nickases” can be used to nick a single strand at the targetsite. Paired nickases can also be used, e.g., to improve specificity,each directed by a pair of different gRNAs targeting sequences. In stillother embodiments, catalytically inactive Cas9 can be fused to aheterologous effector domain, such as a transcriptional repressor, toaffect gene expression.

In general, a CRISPR system is characterized by elements that promotethe formation of a CRISPR complex at the site of the target sequence.Typically, in the context of formation of a CRISPR complex, “targetsequence” generally refers to a sequence to which a guide sequence isdesigned to have complementarity, where hybridization between the targetsequence and a guide sequence promotes the formation of a CRISPRcomplex. Full complementarity is not necessarily required, providedthere is sufficient complementarity to cause hybridization and promoteformation of a CRISPR complex. The target sequence may comprise anypolynucleotide, such as DNA or RNA polynucleotides. Generally, asequence or template that may be used for recombination into thetargeted locus comprising the target sequences is referred to as an“editing template” or “editing polynucleotide” or “editing sequence”. Insome aspects, an exogenous template polynucleotide may be referred to asan editing template. In some aspects, the recombination is homologousrecombination.

In some embodiments, one or more vectors driving expression of one ormore elements of the CRISPR system are introduced into the cell suchthat expression of the elements of the CRISPR system direct formation ofthe CRISPR complex at one or more target sites. For example, a Casenzyme, a guide sequence linked to a tracr-mate sequence, and a tracrsequence could each be operably linked to separate regulatory elementson separate vectors. Alternatively, two or more of the elementsexpressed from the same or different regulatory elements, may becombined in a single vector, with one or more additional vectorsproviding any components of the CRISPR system not included in the firstvector. In some embodiments, CRISPR system elements that are combined ina single vector may be arranged in any suitable orientation. In someembodiments, the CRISPR enzyme, guide sequence, tracr mate sequence, andtracr sequence are operably linked to and expressed from the samepromoter. In some embodiments, a vector comprises a regulatory elementoperably linked to an enzyme-coding sequence encoding the CRISPR enzyme,such as a Cas protein.

In some embodiments, a CRISPR enzyme in combination with (and optionallycomplexed with) a guide sequence is delivered to the cell. Typically,CRISPR/Cas9 technology may be used to knockdown gene expression ofSuv39h1 in the engineered cells. For example, Cas9 nuclease and a guideRNA specific to the Suv39h1 gene can be introduced into cells, forexample, using lentiviral delivery vectors or any of a number of knowndelivery method or vehicle for transfer to cells, such as any of anumber of known methods or vehicles for delivering Cas9 molecules andguide RNAs (see also below).

Delivery of Nucleic Acids Encoding the Gene Disrupting Molecules andComplexes

In some embodiments, a nucleic acid encoding the DNA-targeting molecule,complex, or combination, is administered or introduced to the cell.Typically, viral and non-viral based gene transfer methods can be usedto introduce nucleic acids encoding components of a CRISPR, ZFP, ZFN,TALE, and/or TALEN system to cells in culture.

In some embodiments, the polypeptides are synthesized in situ in thecell as a result of the introduction of polynucleotides encoding thepolypeptides into the cell. In some aspects, the polypeptides could beproduced outside the cell and then introduced thereto.

Methods for introducing a polynucleotide construct into animal cells areknown and include, as non-limiting examples, stable transformationmethods wherein the polynucleotide construct is integrated into thegenome of the cell, transient transformation methods wherein thepolynucleotide construct is not integrated into the genome of the cell,and virus mediated methods.

In some embodiments, the polynucleotides may be introduced into the cellby for example, recombinant viral vectors (e.g. retroviruses,adenoviruses), liposome and the like. Transient transformation methodsinclude microinjection, electroporation, or particle bombardment. Thenucleic acid is administered in the form of an expression vector.Preferably, the expression vector is a retroviral expression vector, anadenoviral expression vector, a DNA plasmid expression vector, or an AAVexpression vector.

Methods of non-viral delivery of nucleic acids include lipofection,nucleofection, microinjection, biolistics, virosomes, liposomes,immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA,artificial virions, and agent-enhanced uptake of DNA. Lipofection isdescribed in e.g., U.S. Pat. Nos. 5,049,386, 4,946,787; and 4,897,355)and lipofection reagents are sold commercially (e.g., Transfectam™ andLipofectin™). Cationic and neutral lipids that are suitable forefficient receptor-recognition lipofection of polynucleotides includethose of Feigner, WO 91/17424; WO 91/16024. Delivery can be to cells(e.g. in vitro or ex vivo administration) or target tissues (e.g. invivo administration).

RNA or DNA viral-based systems include retroviral, lentivirus,adenoviral, adeno-associated and herpes simplex virus vectors for genetransfer.

For a review of gene therapy procedures, see Anderson, Science256:808-813 (1992); Nabel & Feigner, TIBTECH 11:211-217 (1993); Mitani &Caskey, TIBTECH 11: 162-166 (1993); Dillon. TIBTECH 11: 167-175 (1993);Miller, Nature 357:455-460 (1992); Van Brunt, Biotechnology 6(10):1149-1154 (1988); Vigne, Restorative Neurology and Neuroscience 8:35-36(1995); Kremer & Perricaudet, British Medical Bulletin 51(1):31-44(1995); Haddada et al., in Current Topics in Microbiology and ImmunologyDoerfler and Bohm (eds) (1995); and Yu et al., Gene Therapy 1: 13-26(1994).

A reporter gene which includes but is not limited toglutathione-5-transferase (GST), horseradish peroxidase (HRP),chloramphenicol acetyltransferase (CAT) beta-galactosidase,beta-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed,DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP),and autofluorescent proteins including blue fluorescent protein (BFP),may be introduced into the cell to encode a gene product which serves asa marker by which to measure the alteration or modification ofexpression of the gene product.

Cell Preparation

Isolation of the cells includes one or more preparation and/ornon-affinity based cell separation steps according to well-knowntechniques in the field. In some examples, cells are washed,centrifuged, and/or incubated in the presence of one or more reagents,for example, to remove unwanted components, enrich for desiredcomponents, lyse or remove cells sensitive to particular reagents. Insome examples, cells are separated based on one or more property, suchas density, adherent properties, size, sensitivity and/or resistance toparticular components.

In some embodiments, the cell preparation includes steps for freezing,e.g., cryopreserving, the cells, either before or after isolation,incubation, and/or engineering. Any of a variety of known freezingsolutions and parameters in some aspects may be used.

Typically, the cells are incubated prior to or in connection withgenetic engineering and/or Suv39h1 inhibition.

The incubation steps can comprise culture, incubation, stimulation,activation, expansion and/or propagation.

Inhibition of Suv39h1 as per the invention may also be achieved in vivoafter injection f the cells to the targeted patients. Typicallyinhibition of suv39h1 is performed using pharmacological inhibitors aspreviously described.

Inhibition of Suv39h1 as per the method as previously described can alsobe performed during stimulation, activation and/or expansion steps. Forexample, PBMCs, or purified T cells, or purified NK cells, or purifiedlymphoid progenitors, are expanded in vitro in presence of thepharmacological inhibitors of Suv39h1 before adoptive transfer topatients. In some embodiments, the compositions or cells are incubatedin the presence of stimulating conditions or a stimulatory agent. Suchconditions include those designed to induce proliferation, expansion,activation, and/or survival of cells in the population, to mimic antigenexposure, and/or to prime the cells for genetic engineering, such as forthe introduction of a genetically engineered antigen receptor.

The incubation conditions can include one or more of particular media,temperature, oxygen content, carbon dioxide content, time, agents, e.g.,nutrients, amino acids, antibiotics, ions, and/or stimulatory factors,such as cytokines, chemokines, antigens, binding partners, fusionproteins, recombinant soluble receptors, and any other agents designedto activate the cells.

In some embodiments, the stimulating conditions or agents include one ormore agent, e.g., ligand, which is capable of activating anintracellular signaling domain of a TCR complex. In some aspects, theagent turns on or initiates TCR/CD3 intracellular signaling cascade in aT cell. Such agents can include antibodies, such as those specific for aTCR component and/or costimulatory receptor, e.g., anti-CD3, anti-CD28,for example, bound to solid support such as a bead, and/or one or morecytokines. Optionally, the expansion method may further comprise thestep of adding anti-CD3 and/or anti CD28 antibody to the culture medium(e.g., at a concentration of at least about 0.5 ng/ml). In someembodiments, the stimulating agents include 1L-2 and/or IL-15, forexample, an IL-2 concentration of at least about 10 units/mL.

In some aspects, incubation is carried out in accordance with techniquessuch as those described in U.S. Pat. No. 6,040,177 to Riddell et al.,Klebanoff et al., J Immunother. 2012; 35(9): 651-660, Terakura et al.,Blood. 2012; 1:72-82, and/or Wang et al. J Immunother. 2012,35(9):689-701.

In some embodiments, the T cells are expanded by adding to theculture-initiating composition feeder cells, such as non-dividingperipheral blood mononuclear cells (PBMC), (e.g., such that theresulting population of cells contains at least about 5, 10, 20, or 40or more PBMC feeder cells for each T lymphocyte in the initialpopulation to be expanded); and incubating the culture (e.g. for a timesufficient to expand the numbers of T cells). In some aspects, thenon-dividing feeder cells can comprise gamma-irradiated PBMC feedercells. In some embodiments, the PBMC are irradiated with gamma rays inthe range of about 3000 to 3600 rads to prevent cell division. In someaspects, the feeder cells are added to culture medium prior to theaddition of the populations of T cells.

In some embodiments, the stimulating conditions include temperaturesuitable for the growth of human T lymphocytes, for example, at leastabout 25 degrees Celsius, generally at least about 30 degrees, andgenerally at or about 37 degrees Celsius.

Optionally, the incubation may further comprise adding non-dividingEBV-transformed lymphoblastoid cells (LCL) as feeder cells. LCL can beirradiated with gamma rays in the range of about 6000 to 10,000 rads.The LCL feeder cells in some aspects is provided in any suitable amount,such as a ratio of LCL feeder cells to initial T lymphocytes of at leastabout 10:1.

In embodiments, antigen-specific T cells, such as antigen-specific CD4+and/or CD8+ T cells, are obtained by stimulating naive or antigenspecific T lymphocytes with antigen. For example, antigen-specific Tcell lines or clones can be generated to cytomegalovirus antigens byisolating T cells from infected subjects and stimulating the cells invitro with the same antigen.

In some aspects, the methods include assessing expression of one or moremarkers on the surface of the engineered cells or cells beingengineered. In one embodiment, the methods include assessing surfaceexpression of one or more target antigen (e.g., antigen recognized bythe genetically engineered antigen receptor) sought to be targeted bythe adoptive cell therapy, for example, by affinity-based detectionmethods such as by flow cytometry.

Vectors and Methods for Cell Genetic Engineering

In some aspects, the genetic engineering involves introduction of anucleic acid encoding the genetically engineered component or othercomponent for introduction into the cell, such as a component encoding agene-disruption protein or nucleic acid.

Generally, the engineering of CARs into immune cells (e.g., T cells)requires that the cells be cultured to allow for transduction andexpansion. The transduction may utilize a variety of methods, but stablegene transfer is required to enable sustained CAR expression in clonallyexpanding and persisting engineered cells.

In some embodiments, gene transfer is accomplished by first stimulatingcell growth, e.g., T cell growth, proliferation, and/or activation,followed by transduction of the activated cells, and expansion inculture to numbers sufficient for clinical applications.

Various methods for the introduction of genetically engineeredcomponents, e.g., antigen receptors, e.g., CARs, are well known and maybe used with the provided methods and compositions. Exemplary methodsinclude those for transfer of nucleic acids encoding the receptors,including via viral, e.g., retroviral or lentiviral, transduction,transposons, and electroporation.

In some embodiments, recombinant nucleic acids are transferred intocells using recombinant infectious virus particles, such as, e.g.,vectors derived from simian virus 40 (SV40), adenoviruses,adeno-associated virus (AAV). In some embodiments, recombinant nucleicacids are transferred into T cells using recombinant lentiviral vectorsor retroviral vectors, such as gamma-retroviral vectors (see, e.g.,Koste et al. (2014) Gene Therapy 2014 Apr. 3.; Carlens et al. (2000) ExpHematol 28(10): 1137-46; Alonso-Camino et al. (2013) Mol Ther Nucl Acids2, e93; Park et al., Trends Biotechnol. 2011 November; 29(11): 550-557.

In some embodiments, the retroviral vector has a long terminal repeatsequence (LTR), e.g., a retroviral vector derived from the Moloneymurine leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV),murine embryonic stem cell virus (MESV), murine stem cell virus (MSCV),spleen focus forming virus (SFFV), or adeno-associated virus (AAV). Mostretroviral vectors are derived from murine retroviruses. In someembodiments, the retroviruses include those derived from any avian ormammalian cell source. The retroviruses typically are amphotropic,meaning that they are capable of infecting host cells of severalspecies, including humans. In one embodiment, the gene to be expressedreplaces the retroviral gag, pol and/or env sequences. A number ofillustrative retroviral systems have been described (e.g., U.S. Pat.Nos. 5,219,740; 6,207,453; 5,219,740; Miller and Rosman (1989)BioTechniques 7:980-990; Miller, A. D. (1990) Human Gene Therapy 1:5-14;Scarpa et al. (1991) Virology 180:849-852; Burns et al. (1993) Proc.Natl. Acad. Sci. USA 90:8033-8037; and Boris-Lawrie and Temin (1993)Cur. Opin. Genet. Develop. 3: 102-109.

Methods of lentiviral transduction are also known. Exemplary methods aredescribed in, e.g., Wang et al. (2012) J. Immunother. 35(9): 689-701;Cooper et al. (2003) Blood. 101: 1637-1644; Verhoeyen et al. (2009)Methods Mol Biol. 506: 97-114; and Cavalieri et al. (2003) Blood.102(2): 497-505.

In some embodiments, recombinant nucleic acids are transferred into Tcells via electroporation {see, e.g., Chicaybam et al, (2013) PLoS ONE8(3): e60298 and Van Tedeloo et al. (2000) Gene Therapy 7(16):1431-1437). In some embodiments, recombinant nucleic acids aretransferred into T cells via transposition (see, e.g., Manuri et al.(2010) Hum Gene Ther 21(4): 427-437; Sharma et al. (2013) Molec TherNucl Acids 2, e74; and Huang et al. (2009) Methods Mol Biol 506:115-126). Other methods of introducing and expressing genetic materialin immune cells include calcium phosphate transfection (e.g., asdescribed in Current Protocols in Molecular Biology, John Wiley & Sons,New York. N.Y.), protoplast fusion, cationic liposome-mediatedtransfection; tungsten particle-facilitated microparticle bombardment(Johnston, Nature, 346: 776-777 (1990)); and strontium phosphate DNAco-precipitation (Brash et al., Mol. Cell Biol., 7: 2031-2034 (1987)).

Other approaches and vectors for transfer of the genetically engineerednucleic acids encoding the genetically engineered products are thosedescribed, e.g., in international patent application, Publication No.:WO2014055668, and U.S. Pat. No. 7,446,190.

Composition of the Invention

The present invention also includes compositions containing the cells asdescribed herein and/or produced by the provided methods. Typically,said compositions are pharmaceutical compositions and formulations foradministration, such as for adoptive cell therapy.

A pharmaceutical composition of the invention generally comprises atleast one engineered immune cell of the invention and a pharmaceuticallyacceptable carrier.

As used herein the language “pharmaceutically acceptable carrier”includes saline, solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents, and thelike, compatible with pharmaceutical administration. Supplementaryactive compounds can further be incorporated into the compositions. Insome aspects, the choice of carrier in the pharmaceutical composition isdetermined in part by the particular engineered CAR or TCR, vector, orcells expressing the CAR or TCR, as well as by the particular methodused to administer the vector or host cells expressing the CAR.Accordingly, there are a variety of suitable formulations. For example,the pharmaceutical composition can contain preservatives. Suitablepreservatives may include, for example, methylparaben, propylparaben,sodium benzoate, and benzalkonium chloride. In some aspects, a mixtureof two or more preservatives is used. The preservative or mixturesthereof are typically present in an amount of about 0.0001 to about 2%by weight of the total composition.

A pharmaceutical composition is formulated to be compatible with itsintended route of administration.

Therapeutic Methods

The present invention also relates to the cells as previously definedfor their use in adoptive therapy (notably adoptive T cell therapy),typically in the treatment of cancer in a subject in need thereof.

Treatment”, or “treating” as used herein, is defined as the applicationor administration of cells as per the invention or of a compositioncomprising the cells to a patient in need thereof with the purpose tocure, heal, alleviate, relieve, alter, remedy, ameliorate, improve oraffect the disease such as cancer, or any symptom of the disease (e.g.,cancer). In particular, the terms “treat’ or treatment” refers toreducing or alleviating at least one adverse clinical symptom associatedwith the disease such as the cancer cancer, e.g., pain, swelling, lowblood count etc.

With reference to cancer treatment, the term “treat’ or treatment” alsorefers to slowing or reversing the progression neoplastic uncontrolledcell multiplication, i.e. shrinking existing tumors and/or halting tumorgrowth. The term “treat’ or treatment” also refers to inducing apoptosisin cancer or tumor cells in the subject.

The subject of the invention (i.e. patient) is a mammal, typically aprimate, such as a human. In some embodiments, the primate is a monkeyor an ape. The subject can be male or female and can be any suitableage, including infant, juvenile, adolescent, adult, and geriatricsubjects. In some embodiments, the subject is a non-primate mammal, suchas a rodent. In some examples, the patient or subject is a validatedanimal model for disease, adoptive cell therapy, and/or for assessingtoxic outcomes such as cytokine release syndrome (CRS). In someembodiments of the invention, said subject has a cancer, is at risk ofhaving a cancer, or is in remission of a cancer.

The cancer may be a solid cancer or a “liquid tumor” such as cancersaffecting the blood, bone marrow and lymphoid system, also known astumors of the hematopoietic and lymphoid tissues, which notably includeleukemia and lymphoma. Liquid tumors include for example acutemyelogenous leukemia (AML), chronic myelogenous leukemia (CML), acutelymphocytic leukemia (ALL), and chronic lymphocytic leukemia (CLL),(including various lymphomas such as mantle cell lymphoma, non-Hodgkinslymphoma (NHL), adenoma, squamous cell carcinoma, laryngeal carcinoma,gallbladder and bile duct cancers, cancers of the retina such asretinoblastoma).

Solid cancers notably include cancers affecting one of the organsselected from the group consisting of colon, rectum, skin, endometrium,lung (including non-small cell lung carcinoma), uterus, bones (such asOsteosarcoma, Chondrosarcomas, Ewing's sarcoma, Fibrosarcomas, Giantcell tumors, Adamantinomas, and Chordomas), liver, kidney, esophagus,stomach, bladder, pancreas, cervix, brain (such as Meningiomas,Glioblastomas, Lower-Grade Astrocytomas, Oligodendrocytomas, PituitaryTumors, Schwannomas, and Metastatic brain cancers), ovary, breast, headand neck region, testis, prostate and the thyroid gland.

Preferably, a cancer according to the invention is a cancer affectingthe blood, bone marrow and lymphoid system as described above. Typicallythe cancer is, or is associated with, multiple myeloma.

In some embodiments, the subject is suffering from or is at risk of aninfectious disease or condition, such as, but not limited to, viral,retroviral, bacterial, and protozoal infections, immunodeficiency,Cytomegalovirus (CMV), Epstein-Barr virus (EBV), adenovirus, BKpolyomavirus. In some embodiments, the disease or condition is anautoimmune or inflammatory disease or condition, such as arthritis,e.g., rheumatoid arthritis (RA), Type I diabetes, systemic lupuserythematosus (SLE), inflammatory bowel disease, psoriasis, scleroderma,autoimmune thyroid disease, Grave's disease, Crohn's disease multiplesclerosis, asthma, and/or a disease or condition associated withtransplant

The present invention also relates to a method of treatment and notablyan adoptive cell therapy, preferably an adoptive T cell therapy,comprising the administration to a subject in need thereof of acomposition a previously described.

In some embodiments, the cells or compositions are administered to thesubject, such as a subject having or at risk for a cancer or any one ofthe diseases as mentioned above. In some aspects, the methods therebytreat, e.g., ameliorate one or more symptom of, the disease orcondition, such as with reference to cancer, by lessening tumor burdenin a cancer expressing an antigen recognized by the engineered cell.

Methods for administration of cells for adoptive cell therapy are knownand may be used in connection with the provided methods andcompositions. For example, adoptive T cell therapy methods aredescribed, e.g., in US Patent Application Publication No. 2003/0170238to Gruenberg et al; U.S. Pat. No. 4,690,915 to Rosenberg; Rosenberg(2011) Nat Rev Clin Oncol. 8(10):577-85). See, e.g., Themeli et al.(2013) Nat Biotechnol. 31(10): 928-933; Tsukahara et al. (2013) BiochemBiophys Res Commun 438(1): 84-9; Davila et al. (2013) PLoS ONE 8(4):e61338.

In some embodiments, the cell therapy, e.g., adoptive cell therapy,e.g., adoptive T cell therapy, is carried out by autologous transfer, inwhich the cells are isolated and/or otherwise prepared from the subjectwho is to receive the cell therapy, or from a sample derived from such asubject. Thus, in some aspects, the cells are derived from a subject,e.g., patient, in need of a treatment and the cells, following isolationand processing are administered to the same subject.

In some embodiments, the cell therapy, e.g., adoptive cell therapy,e.g., adoptive T cell therapy, is carried out by allogeneic transfer, inwhich the cells are isolated and/or otherwise prepared from a subjectother than a subject who is to receive or who ultimately receives thecell therapy, e.g., a first subject. In such embodiments, the cells thenare administered to a different subject, e.g., a second subject, of thesame species. In some embodiments, the first and second subjects aregenetically identical. In some embodiments, the first and secondsubjects are genetically similar. In some embodiments, the secondsubject expresses the same HLA class or supertype as the first subject.

Administration of at least one cell according to the invention to asubject in need thereof may be combined with one or more additionaltherapeutic agents or in connection with another therapeuticintervention, either simultaneously or sequentially in any order. Insome contexts, the cells are co-administered with another therapysufficiently close in time such that the cell populations enhance theeffect of one or more additional therapeutic agents, or vice versa. Insome embodiments, the cell populations are administered prior to the oneor more additional therapeutic agents. In some embodiments, the cellpopulations are administered after to the one or more additionaltherapeutic agents.

With reference to cancer treatment, a combined cancer treatment caninclude but is not limited to chemotherapeutic agents, hormones,anti-angiogens, radiolabelled compounds, immunotherapy, surgery,cryotherapy, and/or radiotherapy.

Immunotherapy includes but is not limited to immune checkpointmodulators (i.e. inhibitors and/or agonists), monoclonal antibodies,cancer vaccines.

Preferably, administration of cell in an adoptive T cell therapyaccording to the invention is combined with administration of immunecheckpoint modulators. Most preferably, the immune checkpoint modulatorscomprise anti-PD-1 and/or anti-PDL-1 inhibitors.

The present invention also relates to the use of a compositioncomprising the engineered immune cell as herein described for themanufacture of a medicament for treating a cancer, an infectious diseaseor condition, an autoimmune disease or condition, or an inflammatorydisease or condition in a subject.

FIGURES

FIG. 1: The number of central memory CD8+ T cells is increased inSuv39h1-deficient mice. A. Representative FACS dot plots of gatedsplenic CD8+ T cells. B. The percentage of central memory CD8+ T cellswas measured in different hematopoietic compartments (black circles,Suv39h1 KO; white circles: WT littermates).

FIG. 2: Stemness and memory signature in endogenous antigen specificSuv39h1 KO CD8+ T cells. Suv39h1-deficient Kb-OVA+ CD8+ T cells have astem cell-like and memory gene expression signature. Gene set enrichmentanalysis (GSEA) is shown.

FIG. 3: Increased survival and self-renewal of Suv39h1 deficient CD8Tcells. A. Experimental set up showing the transfer of congenic CD45.2Kb-OVA pentamers+CD8+ T cells, isolated from WT and Suv39h1 KO LM-OVAinfected mice, into naïve recipient CD45.1 mice (1: CD8+ T subsets sortand transfer (DO); 2: analysis of self-renewal and differentiation(D40-44). 40 days after the adoptive transfer the naïve recipients micewere challenge with LM-OVA, and 4 days later the Kb-OVA pentamers+CD8+ Tcells were analyzed by FACS. B. Representative FACS plots of donor andrecipient CD8+ T cells are shown. C. The total number of recovered CD8+T cells was measured. The results are combined data from 2 independentexperiments.

FIG. 4: Suv39h1 deficient CD8+OT-1 cells control tumor growth. (A)Treated IL-2/OVA Suv39h1-KO CD45.2 OT-1 and littermate WT cells weretransferred i.v. into recipient bearing tumor mice after 7 days ofinoculation and were intraperitoneally injected with anti-PD-1 antibody(Bio X Cell, RMP-14). Tumor growth curves of treated IL-2/OVA littermateWT (B) and Suv39h1-KO OT-1 cells (C) adoptively transferred to recipientbearing tumor mice.

RESULTS

Material and Methods:

Littermate and Suv39h1-KO mice were infected i.v. with 5×10³ CFU andchallenged 40 days later with 2×10⁶ CFU recombinant Listeriamonocytogenes expressing OVA (LM-OVA, derived from wild type strain140403s). The bacteria were grown in TSB medium (BD Bioscience) tillearly log phase and their growth was assessed with a photometer atOD₆₀₀. Naïve, dump⁻ CD44^(high) K^(b)-OVA⁺ CD8⁺ T cells and relatedsubsets were FACS-sorted. For each subset analysed, we have collected 3or 4 biological replicates. RNA was extracted using Rneasy Micro Kit(QUIGEN) according to the manufacturer protocol. A column DNAsetreatment was included (QUIGEN). For each condition, RNA was employed tosynthetize cDNA according to the standard Affymetrix protocol. LabelledDNA was hybridized on the Affymetrix mouse Gene 2.1 ST, and processed onan Affymetrix GeneTitan device.

Microarray Data Analysis:

Microarray data were processed into R (version 3.0.0) using packagesfrom the Bioconductor. Raw data CEL files were used and the qualitycontrol analysis was performed using ArrayQualityMetrics package. Theraw data were preprocessed using the RMA method available in oligopackage. Probes with no annotation were removed from analysis. Moderatedt-tests were performed using the limma package and the p-values wereadjusted using the multiple testing with the Benjamini Hochberg method.Finally, we considered as statistically significant if adjusted p-valueis lower than 5%.

Gene Set Enrichment Analysis.

Gene Set Enrichment Analysis (GSEA) was performed with gene with theimmunologic signatures (C7) and C2 (curated) gene sets from MolecularSignatures DataBase (MSigDB database v5.1,http://software.broadinstitute.org/gse/msigdb/indexjsp). GMT file wasdownloaded with the gene symbol information. The GCT file was composedof a total of 41345 probes and was imported into GSEA. GSEA was runningwith default parameters except the number of permutation (n=10000). TheBubbleGUM analysis has been done as previously described.

Analysis of Tumor Growth inSuv39h1Deficient CD8+OT-1 Cells werePerformed as Follow:

Total spleen and lymph node from male Suv39h1-KO CD45.2 OT-1 (specificfor OVA257-264 peptide SIINFEKL in a H2-Kb MHC class I context) andlittermate WT mice were cultured in complete medium (RPMI 1640supplemented with 10% FBS, penicillin-streptomycin, and L-glutamine) for3 days and then activated with 100 UI/ml IL-2 and 1 ug/ml OVA257-264peptide for other 3 days.

For tumor inoculation, 1×10⁶ EL4-OVA lymphoma cells were injectedsubcutaneously into the right flank of CD45.1/C57BL/6 male mice andafter 7 days, mice were injected i.v. with 2×10⁶ IL-2/OVA treatedSuv39h1-KO CD45.2 OT-1 or littermate WT cells.

Mice were intraperitoneally injected with anti-PD1 (Bio X Cell, RMP-14)administrated at a dose of 7.5 mg/Kg body weight per dose twice/weekduring 2 weeks. Tumor growth was measured using a manual calliper.

Results:

1) Our results show that in the cell population obtained from Suv39h1-KOmice, early progenitors with “stem cell-like” phenotype accumulate andre-program with increased efficiency into longed-lived central memory Tcells expressing both CD44 and CD62L as compared to the cell populationobtained from wild-type mice. As illustrated in FIG. 1, the proportionand numbers of central memory T cells (expressing both CD44 and CD62L)are both increased in Suv39h1-defective mice.

2) Transcriptomic analysis was used to understand the mechanism of thispeculiar phenotype:

Wild-type and Suv39h1-deficient mice were immunized with OVA-expressingListeria m. and OVA-specific T cells were isolated at day 7 afterimmunization using sorting flow cytometry.

After Affymetrix analysis of the cells, we found that effector T cellsfrom Suv39h1 express higher levels of mRNAs coding for stem-cellcell-related proteins (CD8+ T stem cell-like memory signature (86genes): Abcb1a Irf8 Rest Abcb1b Jarid2 Rif1 Alpl Kat6a Rnf138 Antxr2Klf4 Rras Arl4c Ldha Sall4 Atr Ldhb Satb1 Baalc Ldhc Setbpl Basp1 LdhdSetdb1 Bcl6 Lect1 Skil Bub1 Lpin1 Smarcad1 Ccr7 Ly6a Sox2 Ccr9 Ly6eSpon1 Cd27 Map3k8 Stat3 Cxcr6 Mapk12 Tbx3 Dock9 Mcm3ap Tcf3 Dusp9 Mcoln2Tcl1 Eomes Myc Tdgf1 Esrrb Nanog Tert Evl Ncor2 Tigit Fas Nr0b1 Tnfaip2Fgf2 Nr1d2 Tnfrsf1b Fut4 P2ry14 Traf1 Gzmk Pax6 Traf4 Handl Pcgf2 Trib2Hesx1 Plekha5 Txnip Ier3 Podxl Zfp42 Il2rb Pou5f1 Zfx 117r Pou6f1 Zic3Irf4 Prkce).

We concluded that OVA-specific T cells from Suv39h1-deficient micestimulated after Listeria m. infection express a “stem cell”-like mRNAsignature.

3) Suv39h1 Deficient CD8+ T Cells Show Increased Survival andSelf-Renewal In Vivo.

Congenic CD45.2 Kb-OVA pentamers+CD8+ T cells, isolated from WT andSuv39h1 KO LM-OVA infected mice were transferred into naïve recipientCD45.1 mice. 40 days after the adoptive transfer the naïve recipientsmice were challenge with LM-OVA, and 4 days later the Kb-OVApentamers+CD8+ T cells were analyzed by FACS.

As illustrated in FIG. 3, the result show that Suv39h1-deficient cellexpress increased levels of a “stem cell-like” signature. They alsosurvive better and re-populate more efficiently wild type mice afteradoptive transfer.

4) Suv39h1 Deficient CD8+OT-1 Cells Control Tumor Growth In Vivo

FIGS. 4 B and C show that mice adoptively transferred with Suv39h1-KOOT-1 cells and treated with anti-PD1 monoclonal antibody controlledtumor growth better than mice that received a similar treatment but withWT OT-1 cells. Of note, by day 30 after tumor injection 4 out of 5 WTOT-1-transferred mice showed growing tumor, compared to only 1 out of 5mice injected with the Suv39h1-KO OT-1 cells.

CONCLUSIONS

Absence of Suv39h1 activity in CD8+ T cells is associated to a betteranti-tumor efficacy in an adoptive T cell transfer-based therapeuticapproach.

The invention claimed is:
 1. A method for treating a subject sufferingfrom cancer comprising: administering to said subject an engineeredimmune cell, wherein the immune cell is a T cell, NK cell or T cellprogenitor, comprising a genetically engineered antigen receptor and aninactivated or disrupted SUV39H1 gene, wherein inactivation ordisruption of the SUV39H1 gene results in enhanced anti-cancer activityof said immune cell.
 2. The method of claim 1, wherein the engineeredimmune cell is a T cell or T cell progenitor.
 3. The method of claim 1,wherein the immune cell is a CD4+ T cell, or a CD8+ T cell, or a CD4+and CD8+ T cell.
 4. The method of claim 1 wherein the cell comprises asecond genetically engineered antigen receptor that recognizes adifferent antigen.
 5. The method of claim 1, wherein the geneticallyengineered antigen receptor is a T cell receptor (TCR).
 6. The method ofclaim 2, wherein the genetically engineered antigen receptor is a T cellreceptor (TCR).
 7. The method of claim 1, wherein the geneticallyengineered antigen receptor is a chimeric antigen receptor (CAR).
 8. Themethod of claim 2, wherein the genetically engineered antigen receptoris a chimeric antigen receptor (CAR).
 9. The method of claim 1, whereinthe genetically engineered antigen receptor is a CAR comprising (a) anintracellular signaling domain from CD3 zeta chain and (b) one or morecostimulatory domains of 4-1BB, CD28, ICOS, OX40 or DAP10.
 10. Themethod of claim 2, wherein the genetically engineered antigen receptoris a CAR comprising (a) an intracellular signaling domain from CD3 zetachain and (b) one or more costimulatory domains of 4-1BB, CD28, ICOS,OX40 or DAP10.
 11. The method of claim 1, wherein the engineered immunecell is autologous.
 12. The method of claim 1, wherein the engineeredimmune cell is allogeneic.
 13. A method for treating a subject sufferingfrom cancer comprising administering to said subject: (1) an engineeredimmune cell, wherein the immune cell is a T cell, NK cell or T cellprogenitor, comprising a genetically engineered antigen receptor and aninactivated or disrupted SUV39H1 gene, wherein inactivation ordisruption of the SUV39H1 gene results in enhanced anti-cancer activityof said immune cell; and (2) a second cancer therapeutic agent.
 14. Themethod of claim 13, wherein the second cancer therapeutic agent is animmune checkpoint modulator, cancer vaccine, chemotherapeutic oranti-angiogen.
 15. A method for treating a subject suffering from cancercomprising administering to said subject: (1) an engineered immune cell,wherein the immune cell is a T cell, NK cell or T cell progenitor,comprising a genetically engineered antigen receptor and an inactivatedor disrupted SUV39H1 gene, wherein inactivation or disruption of theSUV39H1 gene results in enhanced anti-cancer activity of said immunecell; and (2) an immune checkpoint modulator.
 16. The method of claim 15wherein the immune checkpoint modulator is an inhibitor of PD1, CTLA4,LAG3, BTLA, OX2R, TIM-3, TIGIT, LAIR-1, PGE2 receptor, EP2/4 adenosinereceptor, or A2AR.
 17. The method of claim 15 wherein the immunecheckpoint modulator is an anti-PD-1 inhibitor or anti-PDL-1 inhibitor.18. The method of claim 1, wherein the disrupted SUV39H1 gene comprisesa deletion.